Bacterial efflux pump inhibitors and methods of treating bacterial infections

ABSTRACT

This invention relates to the field of antimicrobial agents and more specifically it relates to Efflux Pump Inhibitor (EPI) compounds to be co-administered with antimicrobial agents for the treatment of infections caused by drug resistant pathogens. The EPI compounds are soft drugs which exhibit a reduced propensity for tissue accumulation. The invention includes novel compounds useful as efflux pump inhibitors, compositions and devices comprising such efflux pump inhibitors, and therapeutic use of such compounds.

RELATED APPLICATION

This application claims priority to U.S. Provisional Application No.60/574,014, filed on May 21, 2004, which is incorporated herein byreference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to the fields of chemistry and medicine. Moreparticular, the invention relates to novel compounds and compositionsand use of compounds as therapeutic agents, including antimicrobialagents and Efflux Pump Inhibitors (EPIs).

2. Description of the Related Art

Antibiotics have been effective tools in the treatment of infectiousdiseases during the last half-century. From the development ofantibiotic therapy to the late 1980s, there was almost complete controlover bacterial infections in developed countries. However, in responseto the pressure of antibiotic usage, multiple resistance mechanisms havebecome widespread and are threatening the clinical utility ofantibacterial therapy. The increase in antibiotic resistant strains hasbeen particularly common in major hospitals and care centers. Theconsequences of the increase in resistant strains include highermorbidity and mortality, longer patient hospitalization, and an increasein treatment costs.

The emergence of resistant bacterial strains is considered to be thenumber one infectious disease threat in hospitals and costs the U.S.Healthcare system $5 billion per year. Infections that were oncetreatable with antibiotics are becoming difficult and in some casesimpossible to treat. As a result, 2 million people in the U.S. areinfected by a bacterial pathogen while in the hospital each year, ofwhich approximately 90,000 die, and the majority incur prolongedhospital and/or outpatient antibiotic therapy at significantpharmacoeconomic cost. More than 70% of the bacteria responsible forthese infections are resistant to at least one of the antibioticscommonly used to fight them. Therefore the prevention of resistance isconsidered to be a high priority among infectious disease clinicians.

Resistance is not an isolated problem for specific antibiotics, but aglobal one affecting all antibiotics. Numerous studies have documented astrong correlation between the increased use of antibiotics and anincrease in bacterial resistance, which can sometimes occur even duringtherapy. For example, one study documented a growth in fluoroquinoloneresistance in Pseudomonas aeruginosa from 25% in 1997 to 33% in 2002.Another study reported that Pseudomonas aeruginosa resistance tofluoroquinolones increased from 29% in 1999 to 38% in 2001, with somehospitals reporting resistance rates as high as 60%. The level offluoroquinolone use is directly correlated to the level of resistance,thus explaining the variability of resistance in different hospitals.Infections caused by resistant strains and or by strains that developresistance during therapy limit the use of what are otherwise safe andeffective fluoroquinolones as first-line therapy in hospitals.

Bacteria have developed several different mechanisms to overcome theaction of antibiotics. These mechanisms of resistance can be specificfor a molecule or a family of antibiotics, or can be non-specific and beinvolved in resistance to unrelated antibiotics. Several mechanisms ofresistance can exist in a single bacterial strain, and those mechanismsmay act independently or they may act synergistically to overcome theaction of an antibiotic or a combination of antibiotics. Specificmechanisms include degradation of the drug, inactivation of the drug byenzymatic modification, and alteration of the drug target. There are,however, more general mechanisms of drug resistance, in which access ofthe antibiotic to the target is prevented or reduced by decreasing thetransport of the antibiotic into the cell or by increasing the efflux ofthe drug from the cell to the outside medium. Both mechanisms can lowerthe concentration of drug at the target site and allow bacterialsurvival in the presence of one or more antibiotics that would otherwiseinhibit or kill the bacterial cells. Some bacteria utilize bothmechanisms, combining a low permeability of the cell wall (includingmembranes) with an active efflux of antibiotics.

In recent years interest in efflux-mediated resistance in bacteria hasbeen triggered by the growing amount of data implicating efflux pumps inclinical isolates. The phenomenon of antibiotic efflux was firstdiscovered in 1980, in the context of the mechanism of tetracyclineresistance in enterobacteria. Since then, it has been shown that effluxof antibiotics can be mediated by more than one pump in a singleorganism and that almost all antibiotics are subject to resistance bythis mechanism.

Some efflux pumps selectively extrude specific antibiotics. Examples ofsuch pumps include the Tet or CmlA transporters, which can extrudetetracycline or chloramphenicol, respectively. Other efflux pumps,so-called multi-drug resistance (MDR) pumps, extrude a variety ofstructurally diverse compounds. In the latter case, a single effluxsystem may confer resistance to multiple antibiotics with differentmodes of action. In this respect, bacterial MDR pumps are similar tomammalian MDR transporters. In fact, one such pump, P-glycoprotein, thefirst discovered MDR pump, confers multiple drug resistance on cancercells and is considered to be one of the major reasons for tumorresistance to anti-cancer therapy. A typical example of bacterial MDRpump is MexAB-OprM from Pseudomonas aeruginosa. This pump has been shownto affect the susceptibility of the organism to almost all antibioticclasses which fluoroquinolones, β-lactams, macrolides, phenicols,tetracyclines, and oxazolidinones.

Efflux pumps in gram-positive bacteria excrete their substrates across asingle cytoplasmic membrane. This is also the case for some pumps ingram-negative bacteria, and as a result their substrates are effluxedinto the periplasmic space. Other efflux pumps from gram-negativebacteria efflux their substrates directly into the external medium,bypassing the periplasm and the outer membrane. These pumps areorganized in complex three component structures, which traverse bothinner and outer membranes. They consist of a transporter located in thecytoplasmic membrane, an outer membrane channel and a periplasmic‘linker’ protein, which brings the other two components into contact. Itis clearly advantageous for gram-negative bacteria to efflux drugs bybypassing the periplasm and outer membrane. In gram-negative bacteriathe outer membrane significantly slows down the entry of both lipophilicand hydrophilic agents. The former, such as erythromycin and fusidicacid, are hindered by the lipopolysaccharide components of the outerleaflet of the outer membrane bilayer. Hydrophilic agents cross theouter membrane through water-filled porins whose size prevents rapiddiffusion, even for small compounds such as fluoroquinolones and someβ-lactams. Thus, direct efflux creates the possibility for two differentmechanisms to work synergistically to provide the cell with a potentdefense mechanism. Furthermore, direct efflux into the medium leads todecreased amounts of drugs not only in the cytoplasmic but also in theperiplasmic space. This could explain the apparently paradoxical findingthat efflux pumps protect gram-negative bacteria from β-lactamantibiotics whose target penicillin-binding proteins are found in theperiplasm.

Many MDR pumps are encoded by the genes, which are normal constituentsof bacterial chromosomes. In this case increased antibiotic resistanceis a consequence of over-expression of these genes. Thus bacteria havethe potential to develop multi-drug resistance without the acquisitionof multiple specific resistance determinants. In some cases, thesimultaneous operation of efflux pumps and other resistance mechanismsin the same cell results in synergistic effects.

While some genes encoding efflux pumps are not expressed in wild typecells and require induction or regulatory mutations for expression tooccur, other efflux genes are expressed constitutively. As a result,wild type cells have basal level of efflux activity. This basal activityof multi-drug efflux pumps in wild type cells contribute to intrinsicantibiotic resistance, or more properly, decreased antibioticsusceptibility. This intrinsic resistance may be low enough for thebacteria to still be clinically susceptible to therapy. However, thebacteria might be even more susceptible if efflux pumps were renderednon-functional, allowing lower doses of antibiotics to be effective. Toillustrate, P. aeruginosa laboratory-derived mutant strain PAM1626,which does not produce any measurable amounts of efflux pump is 8 to 10fold more susceptible to levofloxacin and meropenem than the parentstrain P. aeruginosa PAM1020, which produces the basal level ofMexAB-OprM efflux pump. Were it not for efflux pumps, the spectrum ofactivity of many so-called ‘gram-positive’ antibiotics could be expandedto previously non-susceptible gram-negative species. This can be appliedto ‘narrow-spectrum’ β-lactams, macrolides, lincosamides,streptogramins, rifamycins, fusidic acid, and oxazolidinones—all ofwhich have a potent antibacterial effect against engineered mutantslacking efflux pumps.

It is clear that in many cases, a dramatic effect on the susceptibilityof problematic pathogens would be greatly enhanced if efflux-mediatedresistance were to be nullified. Two approaches to combat the adverseeffects of efflux on the efficacy of antimicrobial agents can beenvisioned: identification of derivatives of known antibiotics that arenot effluxed and development of therapeutic agents that inhibittransport activity of efflux pumps and could be used in combination withexisting antibiotics to increase their potency.

There are several examples when the first approach has been successfullyreduced to practice. These examples include new fluoroquinolones, whichare not affected by multidrug resistance pumps in Staphylococcus aureusor Streptococcus pneumonia or new tetracycline and macrolidederivatives, which are not recognized by the correspondingantibiotic-specific pumps. However, this approach appears to be muchless successful in the case of multidrug resistance pumps fromgram-negative bacteria. In gram-negative bacteria, particularrestrictions are imposed on the structure of successful drugs: they mustbe amphiphilic in order to cross both membranes. It is this veryproperty that makes antibiotics good substrates of multi-drug resistanceefflux pumps from gram-negative bacteria. In the case of these bacteriathe efflux pump inhibitory approach becomes the major strategy inimproving the clinical effectiveness of existing antibacterial therapy.

SUMMARY OF THE INVENTION

One embodiment disclosed herein is a compound having the structure ofFormula (II):

-   -   wherein:    -   L-AA-1 together with attached amine and carbonyl groups is a        natural or artificial α-amino acid residue having an        (S)-configuration, with the proviso that the a-amino        functionality in the (S)-amino acid residue is not a member of a        heterocyclic ring and with the proviso that the compound does        not have the formula:

-   -   D-AA-2 together with attached amine and carbonyl groups is a        natural or artificial α-amino acid residue having an        (R)-configuration;    -   CG-1 is hydrogen or a carbon-linked capping group;    -   CG-2 is a carbon-linked capping group, wherein when CG-1 is a        carbon-linked capping group, CG-1 and CG-2 are optionally linked        together to form a 5- or 6-membered ring; and    -   any amino groups that are not part of an amide group are        optionally acylated with a natural or artificial amino acid        residue having an (S)-configuration. In some embodiments, L-AA-1        comprises an amino group. In some embodiments, D-AA-2 comprises        an amino group. In some embodiments, amino group is a primary        amine. In some embodiments, CG-1 is hydrogen. In some        embodiments, the compound comprises at least two amino groups.        In some embodiments, L-AA-1 is selected from the group        consisting of:

-   -   wherein:    -   X₁ is selected from the group consisting of —O— and —S—;    -   m₁ is an integer from 0 to 4;    -   n₁ is an integer from 0 to 1;    -   R₁₁ is selected from the group consisting of —NH₂, —NH—CH(═NH),        —NH—C(CH₃)(═NH), —CH(═NH)NH₂, and —NH—C(═O—NH)NH₂;    -   each A₅ is separately selected from the group consisting of ═CH—        and ═N—, with the proviso that no more than four A₅ are ═N—;    -   each B₅ is separately selected from the group consisting of        ═CH—, ═N—, —O—, —S—, —NH—, and —N(R₆)—, with the proviso that no        more than three B₅ are heteroatoms;    -   R₆ is selected from the group consisting of hydrogen, C₁₋₆        alkyl, and C₃₋₆ cycloalkyl;    -   R₁₃ is selected from the group consisting of —NH₂, —CH₂NH₂,        —CH₂CH₂NH₂, —OCH₂CH₂NH₂, —NH—CH(═NH), —CH₂NH—CH(═NH),        —NH—C(CH₃)(═NH), —CH₂—NH—C(CH₃)(═NH), —C(═NH)NH₂,        —OCH₂C(═NH)NH₂, —NH—C(═NH)NH₂, and —CH₂NH—C(═NH)NH₂;    -   R₁₄ and R₁₅ are separately selected from the group consisting of        hydrogen, halogen, methyl, ethyl, hydroxyl, hydroxymethyl,        methoxyl, trifluoromethyl, and trifluoromethoxyl;    -   Y₁ is selected from the group consisting of —CH₂—, —O—, and —S—;    -   p₁ is an integer from 0 to 1; and    -   the wavy line with subscript N indicates point of attachment to        the amine group that is attached to L-AA-1 and the wavy line        with subscript C indicates point of attachment to the carbonyl        group that is attached to L-AA-1.

In some embodiments, D-AA-2 is selected from the group consisting of:

-   -   wherein:    -   Ar is an optionally substituted aryl or heteroaryl;    -   R₃₁ is selected from the group consisting of optionally        substituted C₁₋₁₀ alkyl, C₁₋₁₀ alkenyl, C₁₋₁₀ alkynyl, and C₁₋₁₀        cycloalkyl;    -   X₃ is selected from the group consisting of —CH₂—, —C(CH₃)₂—,        —O—, and —S—;    -   m₃ is an integer from 1 to 2; and    -   n₃ is an integer from 0 to 2. In some embodiments, D-AA-1 is        selected from the group consisting of:

-   -   wherein:    -   R₃₂ and R₃₃ are separately selected from the group consisting of        hydrogen, methyl, ethyl, n-propyl, isopropyl, cyclopropyl,        tert-butyl, trifluoromethyl, hydroxyl, hydroxymethyl, methoxyl,        trifluoromethoxyl, and halogen;    -   R₃₄ is selected from the group consisting of hydrogen, methyl,        ethyl, n-propyl, isopropyl, cyclopropyl, tert-butyl,        trifluoromethyl, hydroxyl, hydroxymethyl, methoxyl,        trifluoromethoxyl, halogen, —NH₂, —CH₂NH₂, —CH₂CH₂NH₂,        —OCH₂CH₂NH₂, —NH—CH(═NH), —CH₂NH—CH(═NH), —NH—C(CH₃)(═NH),        —CH₂—NH—C(CH₃)(═NH), —C(═NH)NH₂, —OCH₂C(═NH)NH₂, —NH—C(═NH)NH₂,        and —CH₂NH—C(═NH)NH₂;    -   each A₅ is separately selected from the group consisting of ═CH—        and ═N—, with the proviso that no more than four A₅ are ═N—;    -   each B₅ is separately selected from the group consisting of        ═CH—, ═N—, —O—, —S—, —NH—, and —N(R₆)—, with the proviso that no        more than three B₅ are heteroatoms;    -   R₆ is selected from the group consisting of hydrogen, C₁₋₆        alkyl, and C₃₋₆ cycloalkyl; and    -   the wavy line with subscript N indicates point of attachment to        the amino group that is attached to D-AA-2 and the wavy line        with subscript C indicates point of attachment to the carbonyl        group that is attached to D-AA-2.

In some embodiments, CG-1 is selected from the group consisting ofhydrogen, optionally substituted C₁₋₆ alkyl, and optionally substitutedC₃₋₇ cycloalkyl; and CG-2 is selected from the group consisting of:

-   -   each optionally substituted with methyl, ethyl, n-propyl,        isopropyl, cyclopropyl, cyclopentyl, cyclohexyl, tert-butyl,        hydroxyl, methoxyl, ethoxyl, hydroxymethyl, trifluoromethyl,        trifluoromethoxyl, or halogen moieties;    -   each A₅ is separately selected from the group consisting of ═CH—        and ═N—, with the proviso that each A₅ containing ring contains        no more than four ═N— groups;    -   each B₅ is separately selected from the group consisting of —C═,        —N═, —O—, —S—, —NH—, and —N(R₆)—, with the proviso that each B₅        containing ring contains no more than three heteroatoms;    -   R₆ is selected from the group consisting of hydrogen, C₁₋₆        alkyl, and C₃₋₆ cycloalkyl;    -   R₅₁ is selected from the group consisting of hydrogen, methyl,        ethyl, and cyclopropyl;    -   m₅ is an integer from 1 to 3;    -   n₅ is an integer from 0 to 2; and    -   p₅ is an integer from 0 to 4.

In some embodiments, —N(CG-1)(CG-2) is selected from the groupconsisting of:

In some embodiments, —N(CG-1)(CG-2) is selected from the groupconsisting of:

Another embodiment disclosed herein is a compound having the structureof Formula (III):

-   -   wherein:    -   R₁ is selected from the group consisting of:

-   -   X₁ is selected from the group consisting of —O— and —S—;    -   m₁ is an integer from 0 to 4;    -   n₁ is an integer from 0 to 1;    -   R₁₁ is selected from the group consisting of —NH₂, —NH—CH(═NH),        —NH—C(CH₃)(═NH), —CH(═NH)NH₂, and —NH—C(═NH)NH₂;    -   R₁₃ is selected from the group consisting of —NH₂, —CH₂NH₂,        —CH₂CH₂NH₂, —OCH₂CH₂NH₂, —NH—CH(═NH), —CH₂NH—CH(═NH),        —NH—C(CH₃)(═NH), —CH₂—NH—C(CH₃)(═NH), —C(═NH)NH₂,        —OCH₂C(═NH)NH₂, —NH—C(═NH)NH₂, and —CH₂NH—C(═NH)NH₂;    -   R₁₄ and R₁₅ are separately selected from the group consisting of        hydrogen, halogen, methyl, ethyl, hydroxyl, hydroxymethyl,        methoxyl, trifluoromethyl, and trifluoromethoxyl;    -   Y₁ is selected from the group consisting of —CH₂—, —O—, and —S—;    -   p₁ is an integer from 0 to 1;    -   R₃ is selected from the group consisting of:

-   -   Ar is an optionally substituted aryl or heteroaryl;    -   X₃ is selected from the group consisting of —CH₂—, —C(CH₃)₂—        —O—, and —S—;    -   m₃ is an integer from 1 to 2;    -   n₃ is an integer from 0 to 2;    -   R₃₁ is selected from the group consisting of optionally        substituted C₁₋₁₀ alkyl, C₁₋₁₀ alkenyl, C₁₋₁₀ alkynyl, and C₁₋₁₀        cycloalkyl;    -   R₄ is selected from the group consisting of hydrogen, optionally        substituted C₁₋₁₀ alkyl, and optionally substituted C₃₋₇        cycloalkyl;    -   R₅ is selected from the group consisting of:

-   -   each optionally substituted with methyl, ethyl, n-propyl,        isopropyl, cyclopropyl, tert-butyl, hydroxyl, methoxyl, ethoxyl,        hydroxymethyl, trifluoromethyl, trifluoromethoxyl, or halogen        moieties;    -   each A₅ is separately selected from the group consisting of ═CH—        and ═N—, with the proviso that each A₅ containing ring contains        no more than four ═N— groups;    -   each B₅ is separately selected from the group consisting of —C═,        —N═, —O—, —S—, —NH—, and —N(R₆)—, with the proviso that each B₅        containing ring contains no more than three heteroatoms;    -   R₆ is selected from the group consisting of hydrogen, C₁₋₆        alkyl, and C₃₋₆ cycloalkyl;    -   R₅₁ is selected from the group consisting of hydrogen, methyl,        ethyl, and cyclopropyl;    -   m₅ is an integer from 1 to 3;    -   n₅ is an integer from 0 to 2;    -   p₅ is an integer from 0 to 4;    -   R₄ is optionally bound to R₅ to form a five-membered or        six-membered heterocyclic ring; and    -   any amino groups are optionally acylated with a natural or        artificial amino acid residue having an (S)-configuration;    -   with the proviso that the compound does not have the formula:

In some embodiments, R₃ is selected from the group consisting of:

-   -   wherein:    -   R₃₁ is selected from the group consisting of ethyl, propyl,        isopropyl, isobutyl, tert-butyl, cyclopropyl, cyclobutyl,        cyclopentyl, and cyclohexyl;    -   R₃₂ and R₃₃ are separately selected from the group consisting of        hydrogen, methyl, ethyl, n-propyl, isopropyl, cyclopropyl,        tert-butyl, trifluoromethyl, hydroxyl, hydroxymethyl, methoxyl,        trifluoromethoxyl, and halogen;    -   R₃₄ is selected from the group consisting of hydrogen, methyl,        ethyl, n-propyl, isopropyl, cyclopropyl, tert-butyl,        trifluoromethyl, hydroxyl, hydroxymethyl, methoxyl,        trifluoromethoxyl, halogen, —NH₂, —CH₂NH₂, —CH₂CH₂NH₂,        —OCH₂CH₂NH₂, —NH—CH(═NH), —CH₂NH—CH(═NH), —NH—C(CH₃)(═NH),        —CH₂—NH—C(CH₃)(═NH), —C(═NH)NH₂, —OCH₂C(═NH)NH₂, —NH—C(═NH)NH₂,        and —CH₂NH—C(═NH)NH₂;    -   X₃ is selected from the group consisting of —CH₂—, —C(CH₃)₂—        —O—, and —S—;    -   m₃ is an integer from 1 to 2; and    -   n₃ is an integer from 0 to 2.

In some embodiments, R₄ is bound to R₅ to form a five-membered orsix-membered heterocyclic ring and the compound of formula III isselected from the group consisting of:

In some embodiments, —N(R₄)(R₅) is selected from the group consistingof:

In some embodiments, the compounds described above are selected from thegroup consisting of:

In some embodiments, the amino acid residues optionally acylating one ormore amino groups in the compounds described above are selected from thegroup consisting of alanine, arginine, asparagine, aspartic acid,cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, and valine.

In some embodiments, the acylated compound is selected from the groupconsisting of:

Another embodiment disclosed herein is a pharmaceutical compositioncomprising a compound as described above in an amount effective toinhibit an efflux pump of a microbe.

Another embodiment disclosed herein is a pharmaceutical composition,comprising a compound as described above in combination with anantimicrobial agent.

Another embodiment disclosed herein is a method of treating orpreventing a microbial infection, comprising administering to a subjectsuffering from the microbial infection an amount effective to inhibit anefflux pump of the microbe of a compound described above.

Another embodiment disclosed herein is a method for treating orpreventing growth of antimicrobial-resistant microbes, comprisingcontacting the microbe with a compound described above and anantimicrobial agent.

Another embodiment disclosed herein is a method for treating a subjectwith an efflux pump inhibitor, wherein the subject is susceptible toaccumulating efflux pump inhibitors in tissue, and wherein the methodcomprises selecting for use in such treatment a compound of as describedabove.

Another embodiment disclosed herein is a method for preventing ortreating a bacterial infection in a subject, wherein the bacteriacausing the infection exhibit antibiotic resistance through an effluxpump mechanism, comprising administering to a subject an antibiotic towhich the bacteria are resistant and administering to the subject acompound as described above in conjunction with the antibiotic, whereinthe compound is selected to reduce or eliminate tissue damage due totissue accumulation thereof.

Another embodiment disclosed herein is a compound as described above foruse in treating or preventing a microbial infection.

Another embodiment disclosed herein is a compound as described above incombination with an antimicrobial agent for use in treating orpreventing a microbial infection.

Another embodiment disclosed herein is a method for treating orpreventing a microbial infection, comprising identifying a subject thatis susceptible to accumulation in tissue of a compound of formula IIA:

-   -   administering to the subject a compound of formula II:

-   -   wherein:    -   D-AA-1 together with attached amine and carbonyl groups is a        first natural or artificial α-amino acid residue having an        (R)-configuration;    -   L-AA-1 together with attached amine and carbonyl groups is the        first α-amino acid residue but having an (S)-configuration;    -   D-AA-2 together with attached amine and carbonyl groups is a        second natural or artificial α-amino acid residue having an        (R)-configuration;    -   CG-1 is hydrogen or a carbon-linked capping group; and    -   CG-2 is a carbon-linked capping group, wherein when CG-1 is a        carbon-linked capping group, wherein CG-1 and CG-2 are        optionally linked together to form a 5- or 6-membered ring.

In some embodiments of the above methods, the subject is a mammal. Insome embodiments, the subject is a human. In some embodiments, themicrobe is a bacteria. In some embodiments, the bacteria is selectedfrom the group consisting of Pseudomonas aeruginosa, Pseudomonasfluorescens, Pseudomonas acidovorans, Pseudomonas alcaligenes,Pseudomonas putida, Stenotrophomonas maltophilia, Burkholderia cepacia,Aeromonas hydrophilia, Escherichia coli, Citrobacter freundii,Salmonella typhimurium, Salmonella typhi, Salmonella paratyphi,Salmonella enteritidis, Shigella dysenteriae, Shigella flexneri,Shigella sonnei, Enterobacter cloacae, Enterobacter aerogenes,Klebsiella pneumoniae, Klebsiella oxytoca, Serratia marcescens,Francisella tularensis, Morganella morganii, Proteus mirabilis, Proteusvulgaris, Providencia alcalifaciens, Providencia rettgeri, Providenciastuartii, Acinetobacter calcoaceticus, Acinetobacter haemolyticus,Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis,Yersinia intermedia, Bordetella pertussis, Bordetella parapertussis,Bordetella bronchiseptica, Haemophilus influenzae, Haemophilusparainfluenzae, Haemophilus haemolyticus, Haemophilus parahaemolyticus,Haemophilus ducreyi, Pasteurella multocida, Pasteurella haemolytica,Branhamella catarrhalis, Helicobacter pylori, Campylobacter fetus,Campylobacter jejuni, Campylobacter coli, Borrelia burgdorferi, Vibriocholerae, Vibrio parahaemolyticus, Legionella pneumophila, Listeriamonocytogenes, Neisseria gonorrhoeae, Neisseria meningitidis, Kingella,Moraxella, Gardnerella vaginalis, Bacteroides fragilis, Bacteroidesdistasonis, Bacteroides 3452A homology group, Bacteroides vulgatus,Bacteroides ovalus, Bacteroides thetaiotaomicron, Bacteroides uniformis,Bacteroides eggerthii, and Bacteroides splanchnicus. In someembodiments, the antimicrobial agent is selected from the groupconsisting of the antimicrobial agent is a quinolone, aminoglycoside,beta-lactam, coumermycin, chloramphenical, lipopeptide, glycopeptide,glycylcycline, ketolide, macrolide, oxazolidonone, rifamycin,streptogramin, and tetracycline. In some embodiments, the antimicrobialagent is selected from the group consisting of ciprofloxacin,levofloxacin, moxifloxacin, ofloxacin, gatifloxacin, cinoxacin,gemifloxacin, norfloxacin, lomofloxacin, pefloxacin, garenoxacin,sitafloxacin, and DX-619.

Another embodiment disclosed herein is a method of identifying acompound useful for efflux pump inhibition but not accumulatingsignificantly in tissue, comprising identifying a compound having thestructure of formula IIA that is effective at inhibiting an efflux pump:

-   -   producing a compound of formula II:

-   -   and determining whether the compound of formula II does not        accumulate significantly in tissue;    -   wherein:    -   D-AA-1 together with attached amine and carbonyl groups is a        first natural or artificial α-amino acid residue having an        (R)-configuration;    -   L-AA-1 together with attached amine and carbonyl groups is the        first α-amino acid residue but having an (S)-configuration;    -   D-AA-2 together with attached amine and carbonyl groups is a        second natural or artificial α-amino acid residue having an        (R)-configuration;    -   CG-1 is hydrogen or a carbon-linked capping group; and    -   CG-2 is a carbon-linked capping group, wherein when CG-1 is a        carbon-linked capping group, wherein CG-1 and CG-2 are        optionally linked together to form a 5- or 6-membered ring.

Another embodiment disclosed herein is a method of treating a patient byadministering an efflux pump inhibitor in conjunction with anantibiotic, wherein the efflux pump inhibitor has the structure:

-   -   wherein AA-1 and AA-2 together with attached amine and carbonyl        groups represent a natural or artificial a-amino acid residue,        ascertaining whether reduced cellular accumulation of efflux        pump inhibitor in the patient is desirable, and if so, selecting        the efflux pump inhibitor from those efflux pump inhibitors        having formula II:

-   -   wherein:    -   L-AA-1 is AA-2 having an (S)-configuration;    -   D-AA-2 is AA-2 having an (R)-configuration;    -   CG-1 is hydrogen or a carbon-linked capping group; and    -   CG-2 is a carbon-linked capping group, wherein when CG-1 is a        carbon-linked capping group, wherein CG-1 and CG-2 are        optionally linked together to form a 5- or 6-membered ring.

Another embodiment disclosed herein is the use of a compound of formulaII for the preparation of a medicament for treating or preventing amicrobial infection without the compound accumulating significantly intissue:

-   -   wherein:    -   L-AA-1 is AA-2 having an (S)-configuration;    -   D-AA-2 is AA-2 having an (R)-configuration;    -   CG-1 is hydrogen or a carbon-linked capping group; and    -   CG-2 is a carbon-linked capping group, wherein when CG-1 is a        carbon-linked capping group, wherein CG-1 and CG-2 are        optionally linked together to form a 5- or 6-membered ring.

Another embodiment disclosed herein is the use of a compound of formulaII in combination with an antimicrobial agent for the preparation of amedicament for treating or preventing a microbial infection without thecompound accumulating significantly in tissue:

-   -   wherein:    -   L-AA-1 is AA-2 having an (S)-configuration;    -   D-AA-2 is AA-2 having an (R)-configuration;    -   CG-1 is hydrogen or a carbon-linked capping group; and    -   CG-2 is a carbon-linked capping group, wherein when CG-1 is a        carbon-linked capping group, wherein CG-1 and CG-2 are        optionally linked together to form a 5- or 6-membered ring.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A to 1C are bar graphs depicting the in vitro stability of threestereoisomers of an EPI compound in tissue homegenates.

FIG. 2 is a bar graph depicting in vitro stability of two stereroisomersof an EPI compound in tissue homogenates at different pHs.

FIG. 3 is a graph depicting the disappearance of an EPI compound and theconcomitant appearance of its degradation product.

FIG. 4 is a bar graph comparing the stability of an EPI compound insupernatant and pellet of kidney tissue homogenates.

FIG. 5 is a bar graph comparing the stability of three stereroisomers ofan EPI compound in the supernatant of rat kidney tissue homgenates.

FIG. 6 is a bar graph comparing the stability of two stereroisomers ofan EPI compound in the supernatant of rabbit kidney tissue homgenates.

FIG. 7 is a bar graph comparing the stability of two stereroisomers ofan EPI compound in human kidney tissue.

FIG. 8 is a bar graph depicting the formation of a metabolite of an EPIcompound in human kidney tissue.

FIG. 9 is a bar graph comparing the stability of two stereoisomers of anEPI compound in human serum as a function of time.

FIG. 10 is a bar graph depicting the stability of two stereoisomers ofan EPI compound in Pseudomonas aeruginosa.

FIG. 11 is a graph depicting the pharmacokinetics of four stereoisomersof an EPI compound after IV bolus administration in rats.

FIG. 12 is a graph depicting the pharmacokinetics of four EPI compoundsafter IV infusion administration in rats.

FIG. 13 is a graph depicting the pharmacokinetics of four stereoisomersof an EPI compound in tissue after IV bolus administration in rats.

FIG. 14 is a bar graph depicting tissue levels of two stereoisomers ofan EPI compound after 5-day repeated dosing in rats.

FIG. 15 is a bar graph depicting tissue levels of an EPI compound inmice after IP bolus administration.

FIG. 16 is a graph depicting serum levels of an EPI compound afteradministration of a prodrug of the compound.

FIG. 17 is a graph depicting serum levels of an EPI compound afteradministration of a prodrug of the compound.

FIG. 18 is a graph depicting mouse survival rates after administrationof an EPI compound with various concentrations of levofloxacin.

FIG. 19 is a graph depicting mouse survival rates after administrationof an EPI compound with various concentrations of levofloxacin.

FIG. 20 is a graph depicting P. aeruginosa growth in a mouse model oflung infection after administration of an EPI compound withlevofloxacin.

FIGS. 21A and 21B are graphs depicting P. aeruginosa growth in a mousemodel of lung infection after administration of an EPI compound with andwithout levofloxacin.

FIG. 22 is a graph depicting P. aeruginosa growth in a mouse model oflung infection after administration of a prodrug of an EPI compound withlevofloxacin.

FIG. 23 is a graph depicting P. aeruginosa growth in a mouse thigh modelof P. aeruginosa infection after administration of an EPI compound withlevofloxacin.

FIG. 24 is a graph depicting P. aeruginosa growth in a mouse thigh modelof P. aeruginosa infection after administration of an EPI compound andits prodrug levofloxacin.

FIG. 25 is a graph depicting P. aeruginosa growth in a mouse thigh modelof P. aeruginosa infection after administration of an EPI compound withlevofloxacin.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Certain dipeptide compounds have been shown to be effective efflux pumpinhibitors. For example, some EPI compounds have been shown to beeffective in combination with fluoroquinolones against P. aeruginosa.See U.S. Pat. Nos. 6,114,310 and 6,245,746, which are both incorporatedherein by reference in their entirety. One such compound was shown todecrease the intrinsic resistance to levofloxacin in wild-type strains(>8-fold), in strains over-expressing the efflux pumps (max 128-fold),and in clinical isolates decreasing the MIC₅₀ and MIC₉₀ (16-fold). Amajor beneficial consequence of the inhibition of multiple efflux pumpswas shown to be a dramatic decrease in the frequency of emergence of P.aeruginosa strains with clinically relevant levels of resistance tofluoroquinolones, both in vitro and in in vivo animal models.

While certain dipeptide/diamine compounds have been shown to possessattractive microbiological profiles and significant efficacy in variousanimal models of infection, it is demonstrated herein that some of thesecompounds have a long tissue half-life and upon repeated dosesaccumulate in tissues (e.g., kidney, liver, and lung) to levels thatinduce cellular and organ damage. Previous efforts to identify analogsthat avoid tissue accumulation by systematically varyingphysico-chemical characteristics have failed. Accordingly, in someembodiments, structural modifications of diamine EPIs are provided thatresult in reduced tissue accumulation and potentially reduced toxicityof these promising future therapeutic agents.

In some embodiments, diamine EPI therapeutic agents are structurallymodified in such a way that they retain the desired target affinitywhile their propensity for accumulation in tissue is reduced due tomodification of the compound observed in tissues, e.g. at the site ofaccumulation. In some embodiments, the modification is a degradation ofthe compound, such as hydrolysis. When administered to a patientsuffering from a microbial infection that employs efflux pump(s) as aresistance mechanism, such drugs undergo tissue-selective degradation(i.e., tissue soft drugs) and exhibit a reduced propensity for tissueaccumulation while inhibiting the activity of the resistance effluxpump(s), allowing a co-administrated antimicrobial agent to accumulatein sufficient concentration in the periplasm or cytoplasm of theinfecting microbe to kill or inhibit the growth of the microbe to treatthe infection. Thus, some embodiments relate to a method for treating amicrobial infection whose causative microbe employs an efflux pumpresistance mechanism, comprising contacting the microbial cell with asoft drug efflux pump inhibitor in combination with an antimicrobialagent. In some embodiments, the soft drug efflux pump inhibitorscomprise a dipeptidic structure, as disclosed herein. In someembodiments, the dipeptidic structure includes two amino functionalitiesthat are not part of the peptide bonds in the compound. In someembodiments, the dipeptidic structure includes absolute stereochemistrythat supports the reduced tissue accumulation properties of thecompound. For example, in some embodiments, the dipeptidic structure hasan L,D stereochemistry, where the N-terminal amino acid has an Lconfiguration and the C-terminal amino acid has a D configuration, whichis demonstrated herein to have reduced tissue accumulation propertiesrelative to other stereochemistries (e.g., D,D).

Another embodiment includes a method for prophylactic treatment of amammal. In this method, a soft drug efflux pump inhibitor isadministered to a mammal at risk of a microbial infection, e.g., abacterial infection. In some embodiments, an antimicrobial agent isadministered in combination with or coadministered with the soft drugefflux pump inhibitor.

Another embodiment includes a method of enhancing the antimicrobialactivity of an antimicrobial agent against a microbe, in which such amicrobe is contacted with a soft drug efflux pump inhibitor, and anantibacterial agent.

In still other embodiments, pharmaceutical compositions are providedthat are effective for treatment of an infection of an animal, e.g., amammal, by a microbe, such as a bacterium or a fungus. The compositionincludes a pharmaceutically acceptable carrier and a soft drug effluxpump inhibitor as described herein. The preferred embodiments alsoprovide antimicrobial formulations that include an antimicrobial agent,a soft drug efflux pump inhibitor, and a carrier. In preferredembodiments, the antimicrobial agent is an antibacterial agent.

While the disclosure herein is directed primarily to compounds fortreating microbial infections that employ efflux pump(s) as a resistancemechanism, it is understood that the methods herein described can beuseful in producing soft drugs suitable for treatment of otherindications, wherein it is desirable that the therapeutic agent exhibita reduced propensity for tissue accumulation.

DEFINITIONS

The term “administration” or “administering” refers to a method ofgiving a dosage of an antimicrobial pharmaceutical composition to avertebrate or invertebrate, including a mammal, a bird, a fish, or anamphibian, where the method is, e.g., intrarespiratory, topical, oral,intravenous, intraperitoneal, or intramuscular. The preferred method ofadministration can vary depending on various factors, e.g., thecomponents of the pharmaceutical composition, the site of the potentialor actual bacterial infection, the microbe involved, and the severity ofan actual microbial infection.

A “carrier” or “excipient” is a compound or material used to facilitateadministration of the compound, for example, to increase the solubilityof the compound. Solid carriers include, e.g., starch, lactose,dicalcium phosphate, sucrose, and kaolin. Liquid carriers include, e.g.,sterile water, saline, buffers, non-ionic surfactants, and edible oilssuch as oil, peanut and sesame oils. In addition, various adjuvants suchas are commonly used in the art may be included. These and other suchcompounds are described in the literature, e.g., in the Merck Index,Merck & Company, Rahway, N.J. Considerations for the inclusion ofvarious components in pharmaceutical compositions are described, e.g.,in Gilman et al. (Eds.) (1990); Goodman and Gilman's: ThePharmacological Basis of Therapeutics, 8th Ed. Pergamon Press.

A “diagnostic” as used herein is a compound, method, system, or devicethat assists in the identification and characterization of a health ordisease state. The diagnostic can be used in standard assays as is knownin the art.

The term “efflux pump” refers to a protein assembly that exportssubstrate molecules from the cytoplasm or periplasm of a cell, in anenergy dependent fashion. Thus an efflux pump will typically be locatedin the cytoplasmic membrane of the cell (spanning the cytoplasmicmembrane). In Gram-negative bacteria the pump may span the periplasmicspace and there may also be portion of the efflux pump, which spans theouter membrane.

An “efflux pump inhibitor” (“EPI”) is a compound that specificallyinterferes with the ability of an efflux pump to export its normalsubstrate, or other compounds such as an antibiotic. The inhibitor mayhave intrinsic antimicrobial (e.g., antibacterial) activity of its own,but at least a significant portion of the relevant activity is due tothe efflux pump inhibiting activity.

“High throughput screening” as used herein refers to an assay thatprovides for multiple candidate agents or samples to be screenedsimultaneously. As further described below, examples of such assaysinclude the use of microtiter plates which are especially convenientbecause a large number of assays can be carried out simultaneously,using small amounts of reagents and samples.

The term “subject,” as used herein, refers to an organism to which thecompounds disclosed herein may be administered and upon which themethods disclosed herein may practiced. In some embodiments, the subjectis an animal. In some embodiments, the animal is a mammal. In someembodiments, the mammal is a human.

The term “mammal” is used in its usual biological sense. Thus, itspecifically includes humans, cattle, horses, dogs, and cats, but alsoincludes many other species.

The term “microbial infection” refers to the invasion of the hostorganism, whether the organism is a vertebrate, invertebrate, fish,plant, bird, or mammal, by pathogenic microbes. This includes theexcessive growth of microbes that are normally present in or on the bodyof a mammal or other organism. More generally, a microbial infection canbe any situation in which the presence of a microbial population(s) isdamaging to a host mammal. Thus, a mammal is “suffering” from amicrobial infection when excessive numbers of a microbial population arepresent in or on a mammal's body, or when the effects of the presence ofa microbial population(s) is damaging the cells or other tissue of amammal. Specifically, this description applies to a bacterial infection.The compounds of preferred embodiments are also useful in treatingmicrobial growth or contamination of cell cultures or other media, orinanimate surfaces or objects, and nothing herein should limit thepreferred embodiments only to treatment of higher organisms, except whenexplicitly so specified in the claims.

The term “multidrug resistance pump” refers to an efflux pump that isnot highly specific to a particular antibiotic. The term thus includesbroad substrate pumps (e.g., those that efflux a number of compoundswith varying structural characteristics). These pumps are different frompumps that are highly specific for tetracyclines. Tetracycline effluxpumps are involved in specific resistance to tetracycline in bacteria.However, they do not confer resistance to other antibiotics. The genesfor the tetracycline pump components are found in plasmids inGram-negative as well as in Gram-positive bacteria.

The term “non-accumulating soft drug” refers to a drug with a structuralfeature allowing it to be modified in vivo, e.g. by modificationincluding metabolism, degradation, and hydrolysis. Such soft drugs canexhibit a reduced propensity for tissue accumulation, for example,intralysosomal accumulation, intranuclear accumulation, cytoplasmicaccumulation, and the like.

The term “pharmaceutically acceptable carrier” or “pharmaceuticallyacceptable excipient” includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents and the like. The use of such media and agents forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive ingredient, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients can also be incorporatedinto the compositions.

The term “pharmaceutically acceptable salt” refers to salts that retainthe biological effectiveness and properties of the compounds of thepreferred embodiments and, which are not biologically or otherwiseundesirable. In many cases, the compounds of the preferred embodimentsare capable of forming acid and/or base salts by virtue of the presenceof amino and/or carboxyl groups or groups similar thereto.Pharmaceutically acceptable acid addition salts can be formed withinorganic acids and organic acids. Inorganic acids from which salts canbe derived include, for example, hydrochloric acid, hydrobromic acid,sulfuric acid, nitric acid, phosphoric acid, and the like. Organic acidsfrom which salts can be derived include, for example, acetic acid,propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid,malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid,ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and thelike. Pharmaceutically acceptable base addition salts can be formed withinorganic and organic bases. Inorganic bases from which salts can bederived include, for example, sodium, potassium, lithium, ammonium,calcium, magnesium, iron, zinc, copper, manganese, aluminum, and thelike; particularly preferred are the ammonium, potassium, sodium,calcium and magnesium salts. Organic bases from which salts can bederived include, for example, primary, secondary, and tertiary amines,substituted amines including naturally occurring substituted amines,cyclic amines, basic ion exchange resins, and the like, specificallysuch as isopropylamine, trimethylamine, diethylamine, triethylamine,tripropylamine, and ethanolamine.

“Solvate” refers to the compound formed by the interaction of a solventand a soft drug, a metabolite, or salt thereof. Suitable solvates arepharmaceutically acceptable solvates including hydrates.

In the context of the response of a microbe, such as a bacterium, to anantimicrobial agent, the term “susceptibility” refers to the sensitivityof the microbe for the presence of the antimicrobial agent. So, toincrease the susceptibility means that the microbe will be inhibited bya lower concentration of the antimicrobial agent in the mediumsurrounding the microbial cells. This is equivalent to saying that themicrobe is more sensitive to the antimicrobial agent. In most cases theminimum inhibitory concentration (MIC) of that antimicrobial agent willhave been reduced.

By “therapeutically effective amount” or “pharmaceutically effectiveamount” is meant an amount of an efflux pump inhibitor, or amountsindividually of an efflux pump inhibitor and an antimicrobial agent, asdisclosed in the preferred embodiments, which have a therapeutic effect,which generally refers to the inhibition to some extent of the normalmetabolism of microbial cells causing or contributing to a microbialinfection. The doses of efflux pump inhibitor and antimicrobial agentwhich are useful in combination as a treatment are therapeuticallyeffective amounts. Thus, as used herein, a therapeutically effectiveamount means those amounts of efflux pump inhibitor and antimicrobialagent which, when used in combination, produce the desired therapeuticeffect as judged by clinical trial results and/or model animal infectionstudies. In particular embodiments, the efflux pump inhibitor andantimicrobial agent are combined in pre-determined proportions and thusa therapeutically effective amount would be an amount of thecombination. This amount and the amount of the efflux pump inhibitor andantimicrobial agent individually can be routinely determined by one ofskill in the art, and will vary, depending on several factors, such asthe particular microbial strain involved and the particular efflux pumpinhibitor and antimicrobial agent used. This amount can further dependupon the patient's height, weight, sex, age and medical history. Forprophylactic treatments, a therapeutically effective amount is thatamount which would be effective if a microbial infection existed.

A therapeutic effect relieves, to some extent, one or more of thesymptoms of the infection, and includes curing an infection. “Curing”means that the symptoms of active infection are eliminated, includingthe elimination of excessive members of viable microbe of those involvedin the infection. However, certain long-term or permanent effects of theinfection may exist even after a cure is obtained (such as extensivetissue damage).

“Treat,” “treatment,” or “treating,” as used herein refers toadministering a pharmaceutical composition for prophylactic and/ortherapeutic purposes. The term “prophylactic treatment” refers totreating a patient who is not yet infected, but who is susceptible to,or otherwise at risk of, a particular infection. The term “therapeutictreatment” refers to administering treatment to a patient alreadysuffering from an infection. Thus, in preferred embodiments, treating isthe administration to a mammal (either for therapeutic or prophylacticpurposes) of therapeutically effective amounts of a soft drug effluxpump inhibitor and an antibacterial (or antimicrobial) agent incombination (either simultaneously or serially).

Soft-Drug Mechanism

As used herein, non-accumulating soft drugs are drugs which arecharacterized by a predictable and controllable intracellular in vivomodification such as by metabolism, preferably intracellular hydrolyticinactivation, to non-accumulating products, preferably after they haveachieved their therapeutic role.

Numerous compounds having useful therapeutic properties possessso-called lysosomotropic character. In general, such properties aredisplayed by molecules bearing one or more basic functionalities with(e.g., amines), such that they are highly protonated at theintralysosomal pH. Such compounds either passively permeate through thelysosomal membrane or are actively transported inside by membrane boundtransport proteins. Having crossed through the membrane, the compoundsencounter the acidic (pH ˜5) environment of the lysosome, become highlyprotonated, and remain entrapped in the intralysosomal space. Theaccumulation of such compounds inside the lysosome can disrupt theproper functioning of the lysosomal enzymatic machinery. Suchaccumulation can also lead to the build-up of osmotic pressure insidethe lysosome, resulting in break-up of the lysosomal membrane followedby leakage of the enzymatic content of the lysosome into the cytoplasm.

Tissues containing cells rich in lysosome vacuoles are particularlysensitive to the toxic effects of intralysosomal accumulation of basicmolecules. Nephrotoxicity due to the intralysosomal accumulation ofbasic molecules in renal tissue is a widely documented phenomenon andhas often been encountered as a serious obstacle to the development ofnew therapeutic agents. The sometimes unwanted accumulation of suchmolecules can occur inside lysosomes or other acidic vacuoles present inother organs (for example, the lung or liver) and can also produce organtoxicity.

One example of tissue accumulation occurs with the drug pentamidine.Pentamidine has been extensively studied mechanistically at the cellularlevel and in animals, as well as clinically in man. The most significantcharacteristic of these cationic compounds is their extensiveaccumulation in a number of body organs, most notably in the kidney andliver, resulting in frank nephrotoxic and hepatotoxic effects. Theincidence of kidney toxicity with parenteral pentamidine inretrospective clinical studies in AIDS patients is above 75%.

Perfused rat kidney studies demonstrate that a combination of mechanisms(filtration, active secretion, and passive reabsorption) are involved inthe renal disposition of pentamidine. Dose proportionality studiesdemonstrated non-linear excretion of pentamidine and significant kidneyaccumulation of the drug. A clear correlation was established betweenextent of kidney sequestration and perturbations in kidney function,most notably GFR, all consistent with observations in the rat thattubulotoxicity of pentamidine is dose-related.

Organic cations are transported in the kidney by the proximal tubule ina multi-step process involving facilitated diffusion and active cationtransport. Once inside the tubular cell, intracellular sequestration canresult in excessive drug concentration, either by binding to cytosolicproteins or accumulating in vesicular compartments (e.g. endosomes andlysosomes). The acidic pH of these organelles can ion-trap cationiccompounds.

Other examples of drugs susceptible to tissue accumulation areaminoglycosides. It has been shown that aminoglycosides accumulate inlysosomes and inhibit lysosomal enzyme activity with concomitantnephrotoxicity.

Thus, the presence of basic functionalities, such as amino groups,within therapeutic compounds can promote unwanted and potentially toxictissue accumulation. However, many biologically active molecules requirebasic functionalities (sometimes multiple basic functionalities permolecule) in order to exert their therapeutic effect through theinteraction with their corresponding molecular targets. Accordingly, ageneral method allowing the modification of otherwise promisingtherapeutic agents in such a way that they retain the desired targetaffinity while exhibiting a reduced propensity for intralysosomalaccumulation or accumulation in other tissue compartments represents anextremely useful tool aiding in the design of new therapeutic moleculesand as such, is highly desirable. To this end, some embodiments providecompounds having basic functionalities, wherein the stereochemistry ofthe compounds has been tailored to reduce tissue accumulation.

Lysosomes are known to contain a fairly large number (about sixty) ofenzymes, most of them having protease activity. Since many of suchenzymes are not found in appreciable amounts outside lysosomes, thespecificity of the proteolytic activity of these enzymes offers thepotential for detoxification of lysosomotropic agents, provided that thelysosomotropic agent contains functionalities which can be cleaved bythese enzymes. The resulting breakdown products of the lysosomotropicagent can potentially exhibit a largely reduced tendency for thedisruption of the lysosomal function. The lysosomotropic agent may alsonot remain trapped inside the lysosome but may, like many smallfragments resulting from, e.g., intralysosomal proteolysis, be removedfrom the lysosome by the active efflux mechanism. Certain enzymes in thelysosomes exhibit esterase activity, which can also offer the potentialfor detoxification of lysosomotropic agents.

An important factor in enzyme activity, such as the proteolytic activityof many of the lysosomal proteolytic enzymes is the pH required fortheir optimal activity. Their catalytic activity displayed at the normallysosomal pH is often largely diminished or completely absent when thepH is increased by one or two pH units.

When the therapeutic agent, such as a drug molecule, is susceptible tothe metabolic proteolytic or esterase clearance it is usually theenzymatic activity of serum, organ specific interstitial fluid, orcytosol that is responsible for the degradation of the drug molecule.Extensive degradation of the drug molecule by such enzymes can result inunacceptably high clearance and reduces the amount of drug below thetherapeutically useful level. On the other hand, since the portion ofthe drug extracted into the lysosomes is typically quite small, thelysosomal enzymes participate only marginally in the metabolic clearanceof therapeutic agents. Therefore, to use the detoxification mechanismdescribed above, it may be desirable that the therapeutic agent moleculecontains a functionality which is not sensitive to the proteolyticactivity of the non-lysosomal enzymes, but which is susceptible to theenzyme or enzymes present inside the lysosome. This allows alysosomotropic drug to exhibit a desired pharmacokinetic property, whileat the same time the portion of the drug that is extracted into thelysosome undergoes detoxification by the specific activity of thelysosomal enzyme or enzymes.

While not being bound to any particular theory, it is likely thatnon-soft diamine EPI compounds undergo intralysosomal accumulation. Thecationic character of diamine EPIs make them similar to pentamidine andaminoglycosides for which intralysosomal accumulation has beendemonstrated. In contrast, it is likely that non-accumulating soft-drugEPIs disclosed herein undergo intralysosomal hydrolysis. Support forthis conclusion includes: 1) serum stability and tissue-mediatedhydrolysis of soft EPIs indicate that hydrolysis occurs intracellularly;2) the presence of a peptide bond in these compounds providesusceptibility to proteolysis; 3) eukaryotic cells have 2 major systemsof intracellular proteolytic activities: (a) lysosomal proteases and (b)ATP-dependent and ubiquitin-assisted proteasome peptidases in cytosoland nucleus. While many soft EPIs may lack necessary features forubiquitinated degradation, their hydrolysis is pH-dependent indicatingintralysosomal degradation. The efficiency of degradation wasdramatically increased at acidic pH, which is present inside lysosomes.Lysosomes contain up to 40 acid hydrolasases that are optimally activeat acidic pH and inactive at neutral pH such as is present in cytoplasmand extracellular compartments. 4) formation of the predicted product ofhydrolysis of soft-EPIs is observed, both in vitro and in vivo. 5)localization of proteolytic enzymes in the soluble fraction of tissuehomogenates (as opposed to membrane bound drug metabolizing P450enzymes) suggest that they are located intralysosomally.

Because non-accumulating soft drugs are hydrolyzed intracellularly, theymay undergo organ selective enzymatic hydrolysis, which is used hereinto mean enzymatic hydrolysis that is observed in homogenates preparedfrom tissues of various organs but not in plasma. Using rodents as modelorganisms it was demonstrated that compounds that undergoorgan-selective hydrolysis in vitro do not accumulate in tissues ofthese organs after administration to animals. Thus the ability toundergo organ-selective hydrolysis is advantageous if tissueaccumulation in organs is expected to result in organ toxicity. In thisrespect organ-selective enzymatic hydrolysis might be considered as away to limit organ-specific toxicity.

The presence of organ-selective enzymatic hydrolysis was furthersupported by the fact that compounds that contain potentially labilepeptide bond were stable in serum but unstable in homogenates preparedfrom tissues of several organs (e.g., kidney, liver, and lung). Theexpected product of proteolytic hydrolysis of the model compound wasidentified after bioanalysis in tissue homogenates. The same product wasalso discovered in tissues of animals receiving the parent compound,indicating that the hydrolysis seen in vitro also occurs in vivo.

The principles discussed above, directed to drugs containing peptidebonds that are susceptible to organ-selective hydrolysis, can also beapplied to compounds containing other chemical bonds that may besusceptible to organ-specific hydrolyzing enzymes found in intracellularcompartments such as lysosomes or to compounds that may be modified byother in vivo enzymes. Accordingly, in one embodiment, a method isprovided for avoiding organ-specific toxicity or accumulation of a drugin organs while administering a therapeutic compound, comprisingadministering a compound that is adapted to resist accumulation in oneor more organs via enzymatic modification occurring at the site ofaccumulation. In some embodiments, the enzymatic modification is adegradation of the compound. In some embodiments, the degradation is aselective metabolism. In some embodiments, the selective metabolism is aselective enzymatic hydrolysis. In some embodiments, the organs areselected from the group consisting of kidney, liver, lung, skin, muscle,brain, pancreas, thymus, adrenal glands, thyroid, ovaries, uterus,mammary glands, spleen, heart, testes, seminal vesicles, bones,cartilage, tendons, and ligaments. In some embodiments, the site ofaccumulation is intracellular. In some embodiments, the site ofaccumulation is within the cytoplasm. In some embodiments, the site ofaccumulation is within an intracellular vesicle, such as but not limitedto peroxisomes, endosomes, or lysosomes. In some embodiments, theselective metabolism is facilitated by enzymes, including but notlimited to, oxidases or hydrolases. In some embodiments, the hydrolaseis an acidic hydrolase. In some embodiments, the acidic hydrolase is alipase, carbohydrase, nuclease, or protease. In some embodiments, theprotease is a lysosomal protease.

In some embodiments, the non-accumulating soft drugs disclosed hereinhave the advantages of low accumulation in tissues as compared withenzymatically stable analogs and similar serum pharmacokinetics ofenzymatically stable and unstable drugs resulting in comparable efficacyof the soft-drugs and there stable analogs.

Some embodiments are directed to the design and therapeutic use of softdrugs. In some embodiments, the soft drugs are modified peptide-like orester-containing molecules which exhibit a reduced tendency toaccumulate in tissue. In some embodiments, soft drug properties includea reduced tendency for accumulation in the acidic vacuole-typeorganelles (lysosomes, and the like), but which do not reach highconcentrations inside the vacuole due to active proteolytic or esteraseenzymatic degradation events occurring inside the vacuole. In theabsence of such a degradation mechanism preventing the accumulation ofthe basic compound inside the cells, the close congeners of suchnon-accumulating soft drugs display accumulation that can produce organtoxicity (e.g., nephrotoxicity).

Compounds

In some embodiments, compounds having the structure of Formula II andprodrugs, pharmaceutically acceptable salts, and hydrates thereof areprovided:

-   -   wherein:    -   L-AA-1 together with attached amine and carbonyl groups is a        natural or artificial α-amino acid residue having an        (S)-configuration, with the proviso that the a-amino        functionality in the (S)-amino acid residue is not a member of a        heterocyclic ring and with the proviso that the compound does        not have the formula:

-   -   D-AA-2 together with attached amine and carbonyl groups is a        natural or artificial α-amino acid residue having an        (R)-configuration;    -   CG-1 is hydrogen or a carbon-linked capping group;    -   CG-2 is a carbon-linked capping group, wherein when CG-1 is a        carbon-linked capping group, CG-1 and CG-2 are optionally linked        together to form a 5- or 6-membered ring; and    -   any amino groups are optionally acylated with a natural or        artificial amino acid residue having an (S)-configuration.

In some embodiments, L-AA-1 and/or D-AA-2 comprise amino groups, such asprimary amines. In one embodiment, the compound comprises at least twoamino groups that are not part of an amide group. In one embodiment,CG-1 is hydrogen.

In one embodiment, L-AA-1 is selected from the group consisting of:

-   -   wherein:    -   X₁ is selected from the group consisting of —O— and —S—;    -   m₁ is an integer from 0 to 4;    -   n₁ is an integer from 0 to 1;    -   R₁₁ is selected from the group consisting of —NH₂, —NH—CH(═NH),        —NH—C(CH₃)(═NH), —CH(═NH)NH₂, and —NH—C(═NH)NH₂;    -   each A₅ is separately selected from the group consisting of ═CH—        and ═N—, with the proviso that no more than four A₅ are ═N—;    -   each B₅ is separately selected from the group consisting of        ═CH—, ═N—, —O—, —S—, —NH—, and —N(R₆)—, with the proviso that no        more than three B₅ are heteroatoms;    -   R₆ is selected from the group consisting of hydrogen, C₁₋₆        alkyl, and C₃₋₆ cycloalkyl;    -   R₁₃ is selected from the group consisting of —NH₂, —CH₂NH₂,        —CH₂CH₂NH₂, —OCH₂CH₂NH₂, —NH—CH(═NH), —CH₂NH—CH(═NH),        —NH—C(CH₃)(═NH), —CH₂—NH—C(CH₃)(═NH), —C(═NH)NH₂,        —OCH₂C(═NH)NH₂, —NH—C(═NH)NH₂, and —CH₂NH—C(═NH)NH₂;    -   R₁₄ and R₁₅ are separately selected from the group consisting of        hydrogen, halogen, methyl, ethyl, hydroxyl, hydroxymethyl,        methoxyl, trifluoromethyl, and trifluoromethoxyl;    -   Y₁ is selected from the group consisting of —CH₂—, —O—, and —S—;    -   p₁ is an integer from 0 to 1; and    -   the wavy line with subscript N indicates point of attachment to        the amine group that is attached to L-AA-1 and the wavy line        with subscript C indicates point of attachment to the carbonyl        group that is attached to L-AA-1.

In one embodiment, D-AA-2 is selected from the group consisting of:

-   -   wherein:    -   Ar is an optionally substituted aryl or heteroaryl;    -   R₃₁ is selected from the group consisting of optionally        substituted C₁₋₁₀ alkyl, C₁₋₁₀ alkenyl, C₁₋₁₀ alkynyl, and C₁₋₁₀        cycloalkyl;    -   X₃ is selected from the group consisting of —CH₂—, —C(CH₃)₂—        —O—, and —S—;    -   m₃ is an integer from 1 to 2; and    -   n₃ is an integer from 0 to 2.

In one embodiment, D-AA-1 is selected from the group consisting of:

-   -   wherein:    -   R₃₂ and R₃₃ are separately selected from the group consisting of        hydrogen, methyl, ethyl, n-propyl, isopropyl, cyclopropyl,        tert-butyl, trifluoromethyl, hydroxyl, hydroxymethyl, methoxyl,        trifluoromethoxyl, and halogen;    -   R₃₄ is selected from the group consisting of hydrogen, methyl,        ethyl, n-propyl, isopropyl, cyclopropyl, tert-butyl,        trifluoromethyl, hydroxyl, hydroxymethyl, methoxyl,        trifluoromethoxyl, halogen, —NH₂, —CH₂NH₂, —CH₂CH₂NH₂,        —OCH₂CH₂NH₂, —NH—CH(═NH), —CH₂NH—CH(═NH), —NH—C(CH₃)(═NH),        —CH₂—NH—C(CH₃)(═NH), —C(═NH)NH₂, —OCH₂C(═NH)NH₂, —NH—C(═NH)NH₂,        and —CH₂NH—C(═NH)NH₂;    -   each A₅ is separately selected from the group consisting of ═CH—        and ═N—, with the proviso that no more than four A₅ are ═N—;    -   each B₅ is separately selected from the group consisting of        ═CH—, ═N, —O—, —S—, —NH—, and —N(R₆)—, with the proviso that no        more than three B₅ are heteroatoms;    -   R₆ is selected from the group consisting of hydrogen, C₁₋₆        alkyl, and C₃₋₆ cycloalkyl; and    -   the wavy line with subscript N indicates point of attachment to        the amine group that is attached to D-AA-2 and the wavy line        with subscript C indicates point of attachment to the carbonyl        group that is attached to D-AA-2.

In some embodiments, CG-1 is selected from the group consisting ofhydrogen, optionally substituted C₁₋₆ alkyl, and optionally substitutedC₃₋₇ cycloalkyl and CG-2 is selected from the group consisting of:

each optionally substituted with methyl, ethyl, n-propyl, isopropyl,cyclopropyl, cyclopentyl, cyclohexyl, tert-butyl, hydroxyl, methoxyl,ethoxyl, hydroxymethyl, trifluoromethyl, trifluoromethoxyl, or halogenmoieties;

-   -   wherein:    -   each A₅ is separately selected from the group consisting of ═CH—        and ═N—, with the proviso that each A₅ containing ring contains        no more than four ═N— groups;    -   each B₅ is separately selected from the group consisting of —C═,        —N═, —O—, —S—, —NH—, and —N(R₆)—, with the proviso that each B₅        containing ring contains no more than three heteroatoms;    -   R₆ is selected from the group consisting of hydrogen, C₁₋₆        alkyl, and C₃₋₆ cycloalkyl;    -   R₅₁ is selected from the group consisting of hydrogen, methyl,        ethyl, and cyclopropyl;    -   m₅ is an integer from 1 to 3;    -   n₅ is an integer from 0 to 2; and    -   p₅ is an integer from 0 to 4.

In another embodiment, a compound having the structure of Formula (III),and prodrugs, hydrates, and pharmaceutically acceptable salts thereof isprovided:

-   -   wherein:    -   R₁ is selected from the group consisting of:

-   -   X₁ is selected from the group consisting of —O— and —S—;    -   m₁ is an integer from 0 to 4;    -   n₁ is an integer from 0 to 1;    -   R₁₁ is selected from the group consisting of —NH₂, —NH—CH(═NH),        —NH—C(CH₃)(═NH), —CH(═NH)NH₂, and —NH—C(═NH)NH₂;    -   R₁₃ is selected from the group consisting of —NH₂, —CH₂NH₂,        —CH₂CH₂NH₂, —OCH₂CH₂NH₂, —NH—CH(═NH), —CH₂NH—CH(═NH),        —NH—C(CH₃)(═NH), —CH₂—NH—C(CH₃)(═NH), —C(═NH)NH₂,        —OCH₂C(═NH)NH₂, —NH—C(═NH)NH₂, and —CH₂NH—C(═NH)NH₂;    -   R₁₄ and R₁₅ are separately selected from the group consisting of        hydrogen, halogen, methyl, ethyl, hydroxyl, hydroxymethyl,        methoxyl, trifluoromethyl, and trifluoromethoxyl;    -   Y₁ is selected from the group consisting of —CH₂—, —O—, and —S—;    -   p₁ is an integer from 0 to 1;    -   R₃ is selected from the group consisting of:

-   -   Ar is an optionally substituted aryl or heteroaryl;    -   X₃ is selected from the group consisting of —CH₂—, —C(CH₃)₂—        —O—, and —S—;    -   m₃ is an integer from 1 to 2;    -   n₃ is an integer from 0 to 2;    -   R₃₁ is selected from the group consisting of optionally        substituted C₁₋₁₀ alkyl, C₁₋₁₀ alkenyl, C₁₋₁₀ alkynyl, and C₁₋₁₀        cycloalkyl;    -   R₄ is selected from the group consisting of hydrogen, optionally        substituted C₁₋₆ alkyl, and optionally substituted C₃₋₇        cycloalkyl;    -   R₅ is selected from the group consisting of:

-   -   each optionally substituted with methyl, ethyl, n-propyl,        isopropyl, cyclopropyl, tert-butyl, hydroxyl, methoxyl, ethoxyl,        hydroxymethyl, trifluoromethyl, trifluoromethoxyl, or halogen        moieties;    -   each A₅ is separately selected from the group consisting of ═CH—        and ═N—, with the proviso that each A₅ containing ring contains        no more than four ═N— groups;    -   each B₅ is separately selected from the group consisting of —C═,        —N═, —O—, —S—, —NH—, and —N(R₆)—, with the proviso that each B₅        containing ring contains no more than three heteroatoms;    -   R₆ is selected from the group consisting of hydrogen, C₁₋₆        alkyl, and C₃₋₆ cycloalkyl;    -   R₅₁ is selected from the group consisting of hydrogen, methyl,        ethyl, and cyclopropyl;    -   m₅ is an integer from 1 to 3;    -   n₅ is an integer from 0 to 2;    -   p₅ is an integer from 0 to 4;    -   R₄ is optionally bound to R₅ to form a five-membered or        six-membered heterocyclic ring; and    -   any amino groups are optionally acylated with a natural or        artificial amino acid residue having an (S)-configuration;    -   with the proviso that the compound does not have the formula:

In some embodiments, R₃ is selected from the group consisting of:

-   -   wherein:    -   R₃₁ is selected from the group consisting of ethyl, propyl,        isopropyl, isobutyl, tert-butyl, cyclopropyl, cyclobutyl,        cyclopentyl, and cyclohexyl;    -   R₃₂ and R₃₃ are separately selected from the group consisting of        hydrogen, methyl, ethyl, n-propyl, isopropyl, cyclopropyl,        tert-butyl, trifluoromethyl, hydroxyl, hydroxymethyl, methoxyl,        trifluoromethoxyl, and halogen;    -   R₃₄ is selected from the group consisting of hydrogen, methyl,        ethyl, n-propyl, isopropyl, cyclopropyl, tert-butyl,        trifluoromethyl, hydroxyl, hydroxymethyl, methoxyl,        trifluoromethoxyl, halogen, —NH₂, —CH₂NH₂, —CH₂CH₂NH₂,        —OCH₂CH₂NH₂, —NH—CH(═NH), —CH₂NH—CH(═NH), —NH—C(CH₃)(═NH),        —CH₂—NH—C(CH₃)(═NH), —C(═NH)NH₂, —OCH₂C(═NH)NH₂, —NH—C(═NH)NH₂,        and —CH₂NH—C(═NH)NH₂;    -   X₃ is selected from the group consisting of —CH₂—, —C(CH₃)₂—        —O—, and —S—;    -   m₃ is an integer from 1 to 2; and    -   n₃ is an integer from 0 to 2.

In some embodiments, R₄ is bound to R₅ to form a five-membered orsix-membered heterocyclic ring and the compound of formula III isselected from the group consisting of:

In some embodiments of the compounds of formula II or III,—N(CG-1)(CG-2) or —N(R₄)(R₅) is selected from the group consisting of:

In some of these embodiments, the compound is selected from the groupconsisting of:

In some embodiments, the compound is selected from the group consistingof:

-   -   2-(S)-Amino-N-{4-amino-1-(S)-[3-phenyl-1-(R)-(quinolin-3-ylcarbamoyl)-propylcarbamoyl]-butyl}-succinamic        acid;    -   2-(S),5-Diamino-pentanoic acid        [3-phenyl-1-(R)-(5,6,7,8-tetrahydro-quinolin-3-ylcarbamoyl)-propyl]-amide;    -   2-(S),5-Diamino-pentanoic acid        [3-phenyl-1-(R)-(quinolin-6-ylcarbamoyl)-propyl]-amide;    -   2-(S),5-Diamino-pentanoic acid        [3-(4-fluoro-phenyl)-1-(R)-(quinolin-3-ylcarbamoyl)-propyl]-amide;    -   2-(S),5-Diamino-pentanoic acid        [2-(4-fluoro-phenyl)-1-(R)-(quinolin-3-ylcarbamoyl)-ethyl]-amide;    -   2-(S),5-Diamino-pentanoic acid        [1-(R)-(6-fluoro-quinolin-3-ylcarbamoyl)-3-phenyl-propyl]-amide;    -   2-(S),5-Diamino-pentanoic acid        [1-(R)-([1,6]naphthyridin-3-ylcarbamoyl)-3-phenyl-propyl]-amide;    -   2-(S),5-Diamino-pentanoic acid        [1-(R)-(6-trifluoromethyl-quinolin-3-ylcarbamoyl)-3-phenyl-propyl]-amide;    -   2-(S),4-Diamino-N-[3-phenyl-1-(R)-(quinolin-3-ylcarbamoyl)-propyl]-butyramide;    -   2-(S),6-Diamino-hexanoic acid        [3-phenyl-1-(R)-(quinolin-3-ylcarbamoyl)-propyl]-amide;    -   2-(S),5-Diamino-pentanoic acid        [1-(R)-(methyl-quinolin-3-yl-carbamoyl)-3-phenyl-propyl]-amide;    -   2-(S),5-Diamino-pentanoic acid        [3-phenyl-1-(R)-(quinoxalin-2-ylcarbamoyl)-propyl]-amide;    -   2-(S),5-Diamino-pentanoic acid        [1-(R)-(quinolin-3-ylcarbamoyl)-3-(4-trifluoromethyl-phenyl)-propyl]-amide;    -   2-(S),5-Diamino-pentanoic acid        [1-(R)-(quinolin-3-ylcarbamoyl)-2-(4-trifluoromethyl-phenyl)-ethyl]-amide;    -   2(R)-(2-(S),5-Diamino-pentanoylamino)-5-methyl-hexanoic acid        quinolin-3-ylamide;    -   2-(S),5-Diamino-pentanoic acid        [2-phenyl-1-(R)-(quinolin-3-ylcarbamoyl)-ethyl]-amide;    -   2-(S),5-Diamino-pentanoic acid        [1-(R)-(3,4-dihydro-1H-isoquinoline-2-carbonyl)-3-phenyl-propyl]-amide;    -   2-(S),5-Diamino-pentanoic acid        [1-(R)-(biphenyl-4-ylcarbamoyl)-3-phenyl-propyl]-amide;    -   2-(S),5-Diamino-pentanoic acid        [1-(R)-(biphenyl-3-ylcarbamoyl)-3-phenyl-propyl-amide;    -   2-(S),5-Diamino-pentanoic acid        [1-(R)-(3-methyl-butylcarbamoyl)-3-phenyl-propyl]-amide;    -   2-(S),5-Diamino-pentanoic acid        [3-phenyl-1-(R)-(quinolin-7-ylcarbamoyl)-propyl]-amide;    -   2-(S),5-Diamino-pentanoic acid        [1-(R)-(2-fluoro-5-trifluoromethyl-phenylcarbamoyl)-3-phenyl-propyl]-amide;    -   2-(S),5-Diamino-pentanoic acid        [3-phenyl-1-(R)-(3,4,5-trifluoro-phenylcarbamoyl)-propyl]-amide;    -   2-(S),5-Diamino-pentanoic acid        [3-phenyl-1-(R)-(2,3,4-trifluoro-phenylcarbamoyl)-propyl]-amide;    -   2-(S),5-Diamino-pentanoic acid        [1-(R)-(5-chloro-2-fluoro-phenylcarbamoyl)-3-phenyl-propyl]-amide;    -   2-(S),5-Diamino-pentanoic acid        [1-(R)-(1-methyl-2-oxo-1,2-dihydro-quinolin-3-ylcarbamoyl)-3-phenyl-propyl]-amide;    -   3-(S)-Amino-N-{4-(S)-amino-4-[3-phenyl-1-(R)-(quinolin-3-ylcarbamoyl)-propylcarbamoyl]-butyl}-succinamic        acid;    -   2-(R)-[2-(S)-Amino-3-(2-amino-ethoxy)-propionylamino]-4-phenyl-N-quinolin-3-yl-butyramide;    -   2-(S),5-Diamino-pentanoic acid        [1-(R)-(3,5-dichloro-pyridin-2-ylcarbamoyl)-3-phenyl-propyl]-amide;    -   2-(S),5-Diamino-pentanoic acid        [1-(R)-(5-fluoro-2-hydroxy-phenylcarbamoyl)-3-phenyl-propyl]-amide;    -   2-(S),5-Diamino-pentanoic acid        [1-(R)-(3,5-difluoro-2-hydroxy-phenylcarbamoyl)-3-phenyl-propyl]-amide;    -   2-(S),5-Diamino-pentanoic acid        [1-(R)-(cinnolin-3-ylcarbamoyl)-3-phenyl-propyl]-amide;    -   2-(S),5-Diamino-pentanoic acid        [1-(R)-(2-oxo-1,2-dihydro-quinolin-3-ylcarbamoyl)-3-phenyl-propyl]-amide;    -   2-(S),5-Diamino-pentanoic acid        [1-(R)-(quinolin-3-ylcarbamoyl)-2-(3,4,5-trifluoro-phenyl)-ethyl]-amide;    -   2-(S),5-Diamino-pentanoic acid        [1-(R)-(quinolin-6-ylcarbamoyl)-2-(3,4,5-trifluoro-phenyl)-ethyl]-amide;    -   3-Amino-(S)—N-{4-(S)-(2-(3)-(S)-amino-3-carboxy-propionylamino)-4-[3-phenyl-1-(R)-(quinolin-3-ylcarbamoyl)-propylcarbamoyl]-butyl}-succinamic        acid;    -   2-(S),5-Diamino-pentanoic acid        [1-(R)-(quinolin-6-ylcarbamoyl)-2-(4-trifluoromethyl-phenyl)-ethyl]-amide;    -   5-Amino-2-(S)-methylamino-pentanoic acid        [3-phenyl-1-(R)-(quinolin-3-ylcarbamoyl)-propyl]-amide;    -   2-(S),5-Diamino-pentanoic acid        [1-(R)-(6-fluoro-quinolin-3-ylcarbamoyl)-2-(4-trifluoromethyl-phenyl)-ethyl]-amide;    -   2-(S),4-Diamino-N-[1-(R)-(6-fluoro-quinolin-3-ylcarbamoyl)-2-(4-trifluoromethyl-phenyl)-ethyl]-butyramide;    -   2-(S),5-Bis-formimidoylamino-pentanoic acid        [3-phenyl-1-(R)-(quinolin-3-ylcarbamoyl)-propyl]-amide;    -   2-(S),5-Diamino-pentanoic acid        [3-(4-fluoro-phenyl)-1-(R)-(quinolin-6-ylcarbamoyl)-propyl]-amide;    -   2-(S),5-Diamino-pentanoic acid        [1-(R)-(benzothiazol-6-ylcarbamoyl)-3-phenyl-propyl]-amide;    -   2-(S),5-Diamino-pentanoic acid        [3-phenyl-1-(R)-(quinoxalin-6-ylcarbamoyl)-propyl]-amide;    -   2-(S),5-Diamino-pentanoic acid        [2-(4-fluoro-phenyl)-1-(R)-(quinolin-6-ylcarbamoyl)-ethyl]-amide;    -   2-(S)-Amino-6-methylamino-hexanoic acid        [3-phenyl-1-(R)-(quinolin-3-ylcarbamoyl)-propyl]-amide;    -   2-(S)-Amino-6-dimethylamino-hexanoic acid        [3-phenyl-1-(R)-(quinolin-3-ylcarbamoyl)-propyl]-amide;    -   2-(S),5-Diamino-pentanoic acid        [3-phenyl-1-(R)-(4,5,6,7-tetrahydro-benzothiazol-2-ylcarbamoyl)-propyl]-amide;    -   2,5-Diamino-pentanoic acid        [3-phenyl-1-(R)-(5-phenyl-[1,3,4]thiadiazol-2-ylcarbamoyl)-propyl]-amide;    -   2-(S),5-Diamino-pentanoic acid        [3-phenyl-1-(R)-(4-phenyl-thiazol-2-ylcarbamoyl)-propyl]-amide;    -   2-(S),5-Diamino-pentanoic acid        [3-phenyl-1-(R)-(3-phenyl-[1,2,4]thiadiazol-5-ylcarbamoyl)-propyl]-amide;    -   2-(S),5-Diamino-pentanoic acid        [1-(R)-(5-bromo-thiazol-2-ylcarbamoyl)-3-phenyl-propyl]-amide;    -   2-(S),5-Diamino-pentanoic acid        [3-phenyl-1-(R)-(5-pyridin-3-yl-thiazol-2-ylcarbamoyl)-propyl]-amide;    -   2-(S),5-Diamino-pentanoic acid        [1-(R)-(benzothiazol-2-ylcarbamoyl)-3-phenyl-propyl]-amide;    -   2-(S),5-Diamino-pentanoic acid        [1-(R)-(2′,4′-dimethyl-[4,5′]bithiazolyl-2-ylcarbamoyl)-3-phenyl-propyl]-amide;    -   2-(S),5-Diamino-pentanoic acid        [3-phenyl-1-(R)-(4-[1,2,4]thiadiazol-3-yl-phenylcarbamoyl)-propyl]-amide;    -   2-(S),5-Diamino-pentanoic acid        {3-phenyl-1-(R)-[(3-phenyl-[1,2,4]thiadiazol-5-ylmethyl)-carbamoyl]-propyl}-amide;        and    -   2-(S),4-Diamino-N-[3-phenyl-1-(R)-(quinolin-6-ylcarbamoyl)-propyl]-butyramide.

In other embodiments of the compounds of formulae II or III,—N(CG-1)(CG-2) or —N(R₄)(R₅) is selected from the group consisting of:

In some embodiments, the amino acid residues optionally acylating one ormore amino groups in the compounds of formulae II or III are selectedfrom the group consisting of alanine, arginine, asparagine, asparticacid, cysteine, glutamic acid, glutamine, glycine, histidine,isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,threonine, tryptophan, tyrosine, and valine. In some embodiments, theacylated compound is selected from the group consisting of:

In some embodiments, prodrugs, metabolites, hydrates, andpharmaceutically acceptable salts of the compounds disclosed herein areprovided.

A “prodrug” refers to an agent that is converted into the parent drug invivo. Prodrugs are often useful because, in some situations, they may beeasier to administer than the parent drug. They may, for instance, bebioavailable by oral administration whereas the parent is not. Theprodrug may also have improved solubility in pharmaceutical compositionsover the parent drug. An example, without limitation, of a prodrug wouldbe a compound which is administered as an ester (the “prodrug”) tofacilitate transmittal across a cell membrane where water solubility isdetrimental to mobility but which then is metabolically hydrolyzed tothe carboxylic acid, the active entity, once inside the cell wherewater-solubility is beneficial. A further example of a prodrug might bea short peptide (polyaminoacid) bonded through an amide linkage wherethe peptide is metabolized to reveal the active moiety. Conventionalprocedures for the selection and preparation of suitable prodrugderivatives are described, for example, in Design of Prodrugs, (ed. H.Bundgaard, Elsevier, 1985), which is hereby incorporated herein byreference in its entirety.

The term “pro-drug ester” refers to derivatives of the compoundsdisclosed herein formed by the addition of any of several ester-forminggroups that are hydrolyzed under physiological conditions. Examples ofpro-drug ester groups include pivoyloxymethyl, acetoxymethyl,phthalidyl, indanyl and methoxymethyl, as well as other such groupsknown in the art, including a (5-R-2-oxo-1,3-dioxolen-4-yl)methyl group.Other examples of pro-drug ester groups can be found in, for example, T.Higuchi and V. Stella, in “Pro-drugs as Novel Delivery Systems”, Vol.14, A.C.S. Symposium Series, American Chemical Society (1975); and“Bioreversible Carriers in Drug Design: Theory and Application”, editedby E. B. Roche, Pergamon Press: New York, 14-21 (1987) (providingexamples of esters useful as prodrugs for compounds containing carboxylgroups). Each of the above-mentioned references is herein incorporatedby reference in their entirety.

Metabolites of the compounds disclosed herein include active speciesthat are produced upon introduction of the compounds into the biologicalmilieu.

The term “pharmaceutically acceptable salt” refers to a salt of acompound that does not cause significant irritation to an organism towhich it is administered and does not abrogate the biological activityand properties of the compound. In some embodiments, the salt is an acidaddition salt of the compound. Pharmaceutical salts can be obtained byreacting a compound with inorganic acids such as hydrohalic acid (e.g.,hydrochloric acid or hydrobromic acid), sulfuric acid, nitric acid,phosphoric acid and the like. Pharmaceutical salts can also be obtainedby reacting a compound with an organic acid such as aliphatic oraromatic carboxylic or sulfonic acids, for example acetic, succinic,lactic, malic, tartaric, citric, ascorbic, nicotinic, methanesulfonic,ethanesulfonic, p-toluensulfonic, salicylic or naphthalenesulfonic acid.Pharmaceutical salts can also be obtained by reacting a compound with abase to form a salt such as an ammonium salt, an alkali metal salt, suchas a sodium or a potassium salt, an alkaline earth metal salt, such as acalcium or a magnesium salt, a salt of organic bases such asdicyclohexylamine, N-methyl-D-glucamine, tris(hydroxymethyl)methylamine,C₁-C₇ alkylamine, cyclohexylamine, triethanolamine, ethylenediamine, andsalts with amino acids such as arginine, lysine, and the like.

If the manufacture of pharmaceutical formulations involves intimatemixing of the pharmaceutical excipients and the active ingredient in itssalt form, then it may be desirable to use pharmaceutical excipientswhich are non-basic, that is, either acidic or neutral excipients.

In various embodiments, the compounds disclosed herein can be usedalone, in combination with other compounds disclosed herein, or incombination with one or more other agents active in the therapeuticareas described herein.

The term “halogen atom,” as used herein, means any one of theradio-stable atoms of column 7 of the Periodic Table of the Elements,e.g., fluorine, chlorine, bromine, or iodine, with fluorine and chlorinebeing preferred.

The term “ester” refers to a chemical moiety with formula—(R)_(n)—COOR′, where R and R′ are independently selected from the groupconsisting of alkyl, cycloalkyl, aryl, heteroaryl (bonded through a ringcarbon) and heteroalicyclic (bonded through a ring carbon), and where nis 0 or 1.

An “amide” is a chemical moiety with formula —(R)_(n)—C(O)NHR′ or—(R)_(n)—NHC(O)R′, where R and R′ are independently selected from thegroup consisting of alkyl, cycloalkyl, aryl, heteroaryl (bonded througha ring carbon) and heteroalicyclic (bonded through a ring carbon), andwhere n is 0 or 1. An amide may be a linking amino acid or a peptidemolecule attached to a molecule of the present invention, therebyforming a prodrug.

Any amine, hydroxy, or carboxyl side chain on the compounds of thepresent invention can be esterified or amidified. The procedures andspecific groups to be used to achieve this end are known to those ofskill in the art and can readily be found in reference sources such asGreene and Wuts, Protective Groups in Organic Synthesis, 3^(rd) Ed.,John Wiley & Sons, New York, N.Y., 1999, which is incorporated herein inits entirety.

The term “aromatic” refers to an aromatic group which has at least onering having a conjugated pi electron system and includes bothcarbocyclic aryl (e.g., phenyl) and heterocyclic aryl groups (e.g.,pyridine). The term includes monocyclic or fused-ring polycyclic (i.e.,rings which share adjacent pairs of carbon atoms) groups. The term“carbocyclic” refers to a compound which contains one or more covalentlyclosed ring structures, and that the atoms forming the backbone of thering are all carbon atoms. The term thus distinguishes carbocyclic fromheterocyclic rings in which the ring backbone contains at least one atomwhich is different from carbon. The term “heteroaromatic” refers to anaromatic group which contains at least one heterocyclic ring.

The term “alkyl,” as used herein, means any unbranched or branched,substituted or unsubstituted, saturated hydrocarbon. The alkyl moiety,may be branched, straight chain, or cyclic. The alkyl group may have 1to 20 carbon atoms (whenever it appears herein, a numerical range suchas “1 to 20” refers to each integer in the given range; e.g., “1 to 20carbon atoms” means that the alkyl group may consist of 1 carbon atom, 2carbon atoms, 3 carbon atoms, etc., up to and including 20 carbon atoms,although the present definition also covers the occurrence of the term“alkyl” where no numerical range is designated). The alkyl group mayalso be a medium size alkyl having 1 to 10 carbon atoms. The alkyl groupcould also be a lower alkyl having 1 to 5 carbon atoms. The alkyl groupmay be designated as “C₁-C₄ alkyl” or similar designations. By way ofexample only, “C₁-C₄ alkyl” indicates that there are one to four carbonatoms in the alkyl chain, i.e., the alkyl chain is selected from thegroup consisting of methyl, ethyl, propyl, iso-propyl, n-butyl,iso-butyl, sec-butyl, and t-butyl.

The alkyl group may be substituted or unsubstituted. When substituted,the substituent group(s) is(are) one or more group(s) individually andindependently selected from substituted or unsubstituted alkyl,substituted or unsubstituted alkenyl, substituted or unsubstitutedalkynyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted cylcloalkenyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, substituted or unsubstitutedheteroaryloxy, heterocyclyl, heterocyclooxy, heteroalicyclyl, hydroxyl,substituted or unsubstituted alkoxy, substituted or unsubstitutedaryloxy, acyl, thiol, substituted or unsubstituted thioalkoxy,alkylthio, arylthio, cyano, halo, carbonyl, thiocarbonyl, acylalkyl,acylamino, acyloxy, aminoacyl, aminoacyloxy, oxyacylamino, keto,thioketo, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl,C-amido, N-amido, S-sulfonamido, N-sulfonamido, C-carboxy, O-carboxy,isocyanato, thiocyanato, isothiocyanato, nitro, silyl,trihalomethanesulfonyl, and substituted or unsubstituted amino,including mono- and di-substituted amino groups, and the protectedderivatives thereof, hydroxyamino, alkoxyamino, nitro, —SO-alkyl,—SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl. Typical alkylgroups include, but are in no way limited to, methyl, ethyl, propyl,isopropyl, butyl, isobutyl, tertiary butyl, pentyl, hexyl, ethenyl,propenyl, butenyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, andthe like. Wherever a substituent is described as being “optionallysubstituted” that substitutent may be substituted with one of the abovesubstituents.

In the present context, the term “cycloalkyl” is intended to coverthree-, four-, five-, six-, seven-, and eight- or more membered ringscomprising carbon atoms only. A cycloalkyl can optionally contain one ormore unsaturated bonds situated in such a way, however, that an aromaticpi-electron system does not arise. Some examples of “cycloalkyl” are thecarbocycles cyclopropane, cyclobutane, cyclopentane, cyclopentene,cyclopentadiene, cyclohexane, cyclohexene, 1,3-cyclohexadiene,1,4-cyclohexadiene, cycloheptane, or cycloheptene.

An “alkenyl” moiety refers to a group consisting of at least two carbonatoms and at least one carbon-carbon double bond. An alkenyl may beunbranched or branched, substituted or unsubstituted, unsaturatedhydrocarbon including polyunsaturated hydrocarbons. In some embodiments,the alkenyl is a C₁-C₆ unbranched, mono-unsaturated or di-unsaturated,unsubstituted hydrocarbons. The term “cycloalkenyl” refers to anynon-aromatic hydrocarbon ring, preferably having five to twelve atomscomprising the ring.

An “alkyne” moiety refers to a group consisting of at least two carbonatoms and at least one carbon-carbon triple bond.

Unless otherwise indicated, the substituent “R” appearing by itself andwithout a number designation refers to a substituent selected from thegroup consisting of alkyl, cycloalkyl, aryl, heteroaryl (bonded througha ring carbon) and heteroalicyclyl (bonded through a ring carbon).

The term “alkoxy” refers to any unbranched, or branched, substituted orunsubstituted, saturated or unsaturated ether, with C₁-C₆ unbranched,saturated, unsubstituted ethers being preferred, with methoxy beingpreferred, and also with dimethyl, diethyl, methyl-isobutyl, andmethyl-tert-butyl ethers also being preferred. The term “cycloalkoxy”refers to any non-aromatic hydrocarbon ring, preferably having five totwelve atoms comprising the ring.

An “O-carboxy” group refers to a RC(═O)O— group, where R is as definedherein.

A “C-carboxy” group refers to a —C(═O)OR groups where R is as definedherein.

An “acetyl” group refers to a —C(═O)CH₃, group.

A “trihalomethanesulfonyl” group refers to a X₃CS(═O)₂— group where X isa halogen.

A “cyano” group refers to a —CN group.

An “isocyanato” group refers to a —NCO group.

A “thiocyanato” group refers to a —CNS group.

An “isothiocyanato” group refers to a —NCS group.

A “sulfinyl” group refers to a —S(═O)—R group, with R as defined herein.

A “S-sulfonamido” group refers to a —S(═O)₂NR, group, with R as definedherein.

A “N-sulfonamido” group refers to a RS(═O)₂NH— group with R as definedherein.

A “trihalomethanesulfonamido” group refers to a X₃CS(═O)₂NR— group withX and R as defined herein.

An “O-carbamyl” group refers to a —OC(═O)—NR, group with R as definedherein.

An “N-carbamyl” group refers to a ROC(═O)NH— group, with R as definedherein.

An “O-thiocarbamyl” group refers to a —OC(═S)—NR, group with R asdefined herein.

An “N-thiocarbamyl” group refers to an ROC(═S)NH— group, with R asdefined herein.

A “C-amido” group refers to a —C(═O)—NR₂ group with R as defined herein.

An “N-amido” group refers to a RC(═O)NH— group, with R as definedherein.

The term “perhaloalkyl” refers to an alkyl group where all of thehydrogen atoms are replaced by halogen atoms.

The term “acylalkyl” refers to a RC(═O)R′— group, with R as definedherein, and R′ being a diradical alkylene group. Examples of acylalkyl,without limitation, may include CH₃C(═O)CH₂—, CH₃C(═O)CH₂CH₂—,CH₃CH₂C(═O)CH₂CH₂—, CH₃C(═O)CH₂CH₂CH₂—, and the like.

Unless otherwise indicated, when a substituent is deemed to be“optionally substituted,” it is meant that the substitutent is a groupthat may be substituted with one or more group(s) individually andindependently selected from alkyl, alkenyl, alkynyl, cycloalkyl, aryl,heteroaryl, heteroalicyclic, hydroxyl, alkoxy, aryloxy, mercapto,alkylthio, arylthio, cyano, halo, carbonyl, thiocarbonyl, O-carbamyl,N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido,S-sulfonamido, N-sulfonamido, C-carboxy, O-carboxy, isocyanato,thiocyanato, isothiocyanato, nitro, silyl, trihalomethanesulfonyl, andamino, including mono- and di-substituted amino groups, and theprotected derivatives thereof. The protecting groups that may form theprotective derivatives of the above substituents are known to those ofskill in the art and may be found in references such as Greene and Wuts,above.

The term “heterocyclyl” is intended to mean three-, four-, five-, six-,seven-, and eight- or more membered rings wherein carbon atoms togetherwith from 1 to 3 heteroatoms constitute said ring. A heterocyclyl canoptionally contain one or more unsaturated bonds situated in such a way,however, that an aromatic pi-electron system does not arise. Theheteroatoms are independently selected from oxygen, sulfur, andnitrogen.

A heterocyclyl can further contain one or more carbonyl or thiocarbonylfunctionalities, so as to make the definition include oxo-systems andthio-systems such as lactams, lactones, cyclic imides, cyclicthioimides, cyclic carbamates, and the like.

Heterocyclyl rings can optionally also be fused to aryl rings, such thatthe definition includes bicyclic structures. Typically such fusedheterocyclyl groups share one bond with an optionally substitutedbenzene ring. Examples of benzo-fused heterocyclyl groups include, butare not limited to, benzimidazolidinone, tetrahydroquinoline, andmethylenedioxybenzene ring structures.

Some examples of “heterocyclyls” include, but are not limited to,tetrahydrothiopyran, 4H-pyran, tetrahydropyran, piperidine, 1,3-dioxin,1,3-dioxane, 1,4-dioxin, 1,4-dioxane, piperazine, 1,3-oxathiane,1,4-oxathiin, 1,4-oxathiane, tetrahydro-1,4-thiazine, 2H-1,2-oxazine,maleimide, succinimide, barbituric acid, thiobarbituric acid,dioxopiperazine, hydantoin, dihydrouracil, morpholine, trioxane,hexahydro-1,3,5-triazine, tetrahydrothiophene, tetrahydrofuran,pyrroline, pyrrolidine, pyrrolidone, pyrrolidione, pyrazoline,pyrazolidine, imidazoline, imidazolidine, 1,3-dioxole, 1,3-dioxolane,1,3-dithiole, 1,3-dithiolane, isoxazoline, isoxazolidine, oxazoline,oxazolidine, oxazolidinone, thiazoline, thiazolidine, and1,3-oxathiolane. Binding to the heterocycle can be at the position of aheteroatom or via a carbon atom of the heterocycle, or, for benzo-fusedderivatives, via a carbon of the benzenoid ring.

In the present context the term “aryl” is intended to mean a carbocyclicaromatic ring or ring system. Moreover, the term “aryl” includes fusedring systems wherein at least two aryl rings, or at least one aryl andat least one C₃₋₈-cycloalkyl share at least one chemical bond. Someexamples of “aryl” rings include optionally substituted phenyl,naphthalenyl, phenanthrenyl, anthracenyl, tetralinyl, fluorenyl,indenyl, and indanyl. The term “aryl” relates to aromatic, including,for example, benzenoid groups, connected via one of the ring-formingcarbon atoms, and optionally carrying one or more substituents selectedfrom heterocyclyl, heteroaryl, halo, hydroxy, amino, cyano, nitro,alkylamido, acyl, C₁₋₆ alkoxy, C₁₋₆ alkyl, C₁₋₆ hydroxyalkyl, C₁₋₆aminoalkyl, C₁₋₆ alkylamino, alkylsulfenyl, alkylsulfinyl,alkylsulfonyl, sulfamoyl, or trifluoromethyl. The aryl group can besubstituted at the para and/or meta positions. In other embodiments, thearyl group can be substituted at the ortho position. Representativeexamples of aryl groups include, but are not limited to, phenyl,3-halophenyl, 4-halophenyl, 3-hydroxyphenyl, 4-hydroxyphenyl,3-aminophenyl, 4-aminophenyl, 3-methylphenyl, 4-methylphenyl,3-methoxyphenyl, 4-methoxyphenyl, 4-trifluoromethoxyphenyl3-cyanophenyl, 4-cyanophenyl, dimethylphenyl, naphthyl, hydroxynaphthyl,hydroxymethylphenyl, trifluoromethylphenyl, alkoxyphenyl,4-morpholin-4-ylphenyl, 4-pyrrolidin-1-ylphenyl, 4-pyrazolylphenyl,4-triazolylphenyl, and 4-(2-oxopyrrolidin-1-yl)phenyl.

In the present context, the term “heteroaryl” is intended to mean aheterocyclic aromatic group where one or more carbon atoms in anaromatic ring have been replaced with one or more heteroatoms selectedfrom the group comprising nitrogen, sulfur, phosphorous, and oxygen.

Furthermore, in the present context, the term “heteroaryl” comprisesfused ring systems wherein at least one aryl ring and at least oneheteroaryl ring, at least two heteroaryl rings, at least one heteroarylring and at least one heterocyclyl ring, or at least one heteroaryl ringand at least one cycloalkyl ring share at least one chemical bond.

The term “heteroaryl” is understood to relate to aromatic, C₃₋₈ cyclicgroups further containing one oxygen or sulfur atom or up to fournitrogen atoms, or a combination of one oxygen or sulfur atom with up totwo nitrogen atoms, and their substituted as well as benzo- andpyrido-fused derivatives, for example, connected via one of thering-forming carbon atoms. Heteroaryl groups can carry one or moresubstituents, selected from halo, hydroxy, amino, cyano, nitro,alkylamido, acyl, C₁₋₆-alkoxy, C₁₋₆-alkyl, C₁₋₆-hydroxyalkyl,C₁₋₆-aminoalkyl, C₁₋₆-alkylamino, alkylsulfenyl, alkylsulfinyl,alkylsulfonyl, sulfamoyl, or trifluoromethyl. In some embodiments,heteroaryl groups can be five- and six-membered aromatic heterocyclicsystems carrying 0, 1, or 2 substituents, which can be the same as ordifferent from one another, selected from the list above. Representativeexamples of heteroaryl groups include, but are not limited to,unsubstituted and mono- or di-substituted derivatives of furan,benzofuran, thiophene, benzothiophene, pyrrole, pyridine, indole,oxazole, benzoxazole, isoxazole, benzisoxazole, thiazole, benzothiazole,isothiazole, imidazole, benzimidazole, pyrazole, indazole, tetrazole,quionoline, isoquinoline, pyridazine, pyrimidine, purine and pyrazine,furazan, 1,2,3-oxadiazole, 1,2,3-thiadiazole, 1,2,4-thiadiazole,triazole, benzotriazole, pteridine, phenoxazole, oxadiazole,benzopyrazole, quinolizine, cinnoline, phthalazine, quinazoline, andquinoxaline. In some embodiments, the substituents are halo, hydroxy,cyano, O—C₁₋₆-alkyl, C₁₋₆-alkyl, hydroxy-C₁₋₆-alkyl, andamino-C₁₋₆-alkyl.

Methods of Preparation

The compounds disclosed herein may be synthesized by methods describedbelow, or by modification of these methods. Ways of modifying themethodology include, among others, temperature, solvent, reagents etc.,and will be obvious to those skilled in the art. In general, during anyof the processes for preparation of the compounds disclosed herein, itmay be necessary and/or desirable to protect sensitive or reactivegroups on any of the molecules concerned. This may be achieved by meansof conventional protecting groups, such as those described in ProtectiveGroups in Organic Chemistry (ed. J. F. W. McOmie, Plenum Press, 1973);and Greene & Wuts, Protective Groups in Organic Synthesis, John Wiley &Sons, 1991, which are both hereby incorporated herein by reference intheir entirety. The protecting groups may be removed at a convenientsubsequent stage using methods known from the art. Synthetic chemistrytransformations useful in synthesizing applicable compounds are known inthe art and include e.g. those described in R. Larock, ComprehensiveOrganic Transformations, VCH Publishers, 1989, or L. Paquette, ed.,Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons,1995, which are both hereby incorporated herein by reference in theirentirety.

In the following schemes P, P₁, P₂ are used to denote protecting groupscompatible with the reaction sequences. Protecting groups P, P₁, P₂existing in a specific synthetic scheme may represent differentprotecting groups but may also represent identical protecting groups ifthe synthetic strategy does not require protecting group orthogonality.SS— is used to mark covalent attachment of an intermediate to apolymeric solid support. X denotes a leaving group in amide coupling orin carbon-carbon bond formation. R_(L), R_(D) and R_(LD) denotesubstituents in alfa position of homochiral-L, homochiral-D and racemicα-amino acid functionality respectively. Other abbreviations usedinclude: Boc=t-butoxycarbonyl, OBn=benzyl ester, CBz=carbobenzyloxy, andOSu=N-hydroxysuccinimide ester.

In the following schemes, additional steps maybe required formanipulation of substituents R_(L) and R_(D). For example hydrogenationin the presence of catalyst can be performed of the cyano group in orderto unmask the aminomethyl group.

The general strategies of assembling the peptidic compounds shown beloware meant to be illustrative and are not meant to be limiting in anyway.

The absolute stereochemistry of the chiral centers is represented instructures with customary wedge-bond representations and the chiralcenters of racemic mixtures are represented with customary wavy-linebond representations. Consequently a structure comprising one wedge bondand one wavy-line bond represents a mixture of diastereoisomers.

When the carboxylic acid functionality is activated for the formation ofthe amide bond the active intermediates can be isolated for theconsecutive coupling reaction with the amine or the activation can beperformed in situ without the isolation of the active intermediate. Theactivation of the carboxylic acid functionality can be performed priorto the addition of the amine as well as in the presence of the amineprovided that a proper activating reagent is selected.

Different protecting groups, coupling strategies, deprotection reactionsshown in the schemes below are typical examples of the methods generallyemployed in the synthesis of peptidic bonds (Principles of PeptideSynthesis, 2^(nd) Ed, M. Bodanszky, Spriner-Verlag, Berlin, 1993) andthe methods shown are meant to be illustrative and are not meant to belimiting in any way.

Method A

In some embodiments, compounds disclosed herein are synthesizedaccording to method A above. One specific example of a synthesisaccording to method A is as follows:

A t-Butoxycarbonyl (Boc) group can be used to protect the aminefunctionality as shown above, however any other protective groupcompatible with the reaction sequence can also be used. Activation byformation of N-hydroxysuccinimide ester (step a) is also meant here tobe an illustrative example of an activation strategy and any otheractivation strategy typically employed for the formation of the amidebond can also be used.

Method B

In some embodiments, compounds disclosed herein are synthesizedaccording to Method B above. The aminoacid intermediate containing theR_(D) substituent can be used in an ester form which may facilitatecoupling and purification of the product of step b of Method B. Onespecific example of a synthesis according to Method B is as follows:

The benzyl ester (Bn) used in the step b of the amide bond formationrepresents an example of a carboxylic acid protecting group which iscompatible with the synthetic sequence. Other ester group compatiblewith the synthetic sequence can also be employed.

Method C

In some embodiments, compounds disclosed herein are synthesizedaccording to Method C above. In cases when the desired aminoacidintermediate of D configuration is not easily available in thehomochiral form, the coupling sequence can be preformed using thecorresponding racemic intermediate which is then followed by theattachment of the homochiral aminoacid of L configuration. The resultingdiastereomeric mixture can be separated by chromatography or bycrystallization as shown in step c of the general scheme for Method Cand the further steps of the sequence can be performed with thestereochemically uniform material. One specific example of a synthesisaccording to Method C is as follows:

Method D

Method D can be used as and alternative to the approach described inMethod C for cases when the desired aminoacid intermediate of Dconfiguration is not easily available in the homochiral form. Theracemic intermediate containing the sidechain intended to be thesidechain of the D configuration aminoacid of the desired product iscoupled to the corresponding protected aminoacid of the L configurationas shown in step b and the resulting diastereoisomeric mixture is usedfor the next steps of the sequence. The separation of thediastereoisomeric components is performed at a later step where theseparation by chromatography or by crystallization can be easilyperformed. Isolation of the desired diastereoisomer can be performed instep e or even after final removal of protecting groups.

One specific example of a synthesis according to Method D is as follows:

Method E

Preparation of the desired peptidic product may be performed byattaching the protected aminoacid intermediate to the polymeric solidsupport which can facilitate later coupling reactions and may eliminatethe need for the purification of the intermediate products. The aminofunctionality of the aminoacid of the L configuration can be attached tothe solid support material as shown in step a. The following steps ofdeprotections, activations and amide bond formations can be performed onthe intermediates attached to solid support and the release of theproduct from the solid support can be then performed at the last step ofthe sequence.

One specific example of a synthesis according to Method E is as follows:

The example above shows a sequence in which the amino functionality ofthe L aminoacid intermediate can be attached to the solid supportthrough the carbamate linker similar in character to t-butoxycarbonylprotecting group (T. Redman et al., Mol. Diversity, 4: 191-197, (1998)).After the completion of the assembly of the molecule attached to thesolid support the final deprotection step can remove all protectinggroups present and detach the product from the solid support. Othercleavable linkers attaching the aminoacid to the solid support can bealso employed and may require separate steps for removal of protectinggroups and detaching the product from the solid support.

Method F

α-Aminoacids employed in the synthetic sequences can have in theirsidechains additional amino groups which may also require protection inorder to perform the desired coupling reactions. Protecting groups usedfor these additional amino functionalities maybe identical to theprotecting groups employed for protection of the α-amino functionalitiesabut may also be different.

One specific example of a synthesis according to Method F is as follows:

The above example utilizes t-butoxycarbonyl protecting groups for bothα- and ω-amines of ornithine which can be activated for coupling in thestep a. Deprotection of both amino functionalities can therefore beperformed at the same time in step f.

Method G

In case when the L-aminoacid moiety contains two amino groups, one ofthem can be protected with a typical amino-protecting group while theother one can be used for the attachment to the polymeric solid supportwhich can facilitate later coupling reactions and may eliminate the needfor the purification of the intermediate products. The free aminofunctionality of the monoprotected aminoacid of the L configuration canbe attached to the solid support material as shown in step a. Thefollowing steps of deprotections, activations and amide bond formationscan be performed on the intermediates attached to solid support and therelease of the product from the solid support can be then performed atthe last step of the sequence.

One specific example of a synthesis according to Method G is as follows:

The above example describes the synthetic sequence in which thecharacter of the attachment of the amino functionality is similar incharacter to the t-butoxycarbonyl group used for the protection of thealfa-amino functionality and the deprotection of the amine can beperformed simultaneously with the detachment of the product from thesolid support. The character of the linker and the protecting group mayalso be such that two separate steps maybe required for the deprotectionof the amine functionality detachment of the product from the solidsupport.

Method H

Method H describes a synthetic sequence in which a homochiralN-protected D-α aminoacid intermediate is in the step a converted intothe amide. The resulting amide can be then deprotected and coupled withthe homochiral N-protected D-α-aminoacid and the resulting intermediatecan be than deprotected to provide desired product. An analogoussequence can be also performed with the racemic intermediate containingthe sidechain intended to be the sidechain of the D configurationaminoacid of the desired product. This racemic intermediate can becoupled with the corresponding protected aminoacid of the Lconfiguration analogously to step c and the resulting diastereisomericmixture obtained can be used for next steps of the sequence. Theseparation of the diastereoisomeric components can be performedimmediately following step c or alternatively can be performed at alater step after final removal of protecting groups.

One specific example of a synthesis according to Method H is as follows:

Method I

Homochiral protected aminoacid intermediates necessary for thepreparation of the target peptidic compounds which are the subject ofthis invention can be used when easily available. Homochiral protectedaminoacid intermediates necessary for the preparation of the targetpeptidic can be also prepared by a variety of methods known in theliterature using stereoselective reactions or employing known strategiesfor the separation of enantiomers (Synthesis of Optically Active α-AminoAcids, R M Williams, Pergamon, N.Y., 1989). Alternatively, the desiredprotected aminoacid intermediates can be prepared by a variety ofmethods in form of a racemic mixture and used as such for coupling withthe chomochiral component (e.g. the intermediate containing theaminoacid moiety intended to become a part of the L configurationaminoacid). Method I exemplifies a strategy which can be used for thepreparation of racemic protected aminoacid intermediates containingmoieties intended to become a part of the D configuration aminoacid.

In step a, the Shiff base of a glycine ester can be alkylated with theappropriate alkylating reagent producing a racemic mixture of thedesired protected precursor of the aminoacid of D configuration. In thefollowing steps (b-e) this precursor can be converted into adiastereoisomeric mixture of the final desired product in its protectedform which can be separated by chromatography or by crystallization(step f) in order to isolate the material of the L,D configuration. Theseparation of the desired diastereoisomer can be also performedfollowing the final deprotection step (steps f and g in the reverseorder).

One specific example of a synthesis according to Method I is as follows:

The above example describes the synthetic sequence in which in step athe Shiff base of glycine ethyl ester can be alkylated with asubstituted benzyl bromide. By hydrolysis of both protecting groups andprotection of the amine (steps b,c) a desired protected aminoacid can beobtained in racemic form. After a series of coupling and deprotectionsteps (c-e) the diastereoisomeric mixture can be separated bychromatography or crystallization.

Method J

Method J describes a strategy which can be used for thefunctionalization of the specific amino functionality of the N-terminaldibasic aminoacid. The proper choice of the orthogonal protecting groupsP₁, P₂, P₃ and P₄ can allow removal of one of the three protectinggroups (steps c, e) and attach the additional aminoacid (bearing the L′moiety) to a selected amino group of the dibasic amino acid.

One specific example of a synthesis according to Method J is as follows:

Method K

Method K can be used as an alternative strategy leading toward a similartarget as described in Method J. Orthogonality of the two protectinggroups P₁ and P₂ is sufficient to allow the introduction of theadditional amino acid moiety to a selected amino group of the dibasicaminoacid (steps d and e). Depending on the order of deprotection of theorthogonal protecting groups P₁ and P₂ introduction of the additionalaminoacid moiety can be selectively performed at either of the aminogroups of the dibasic aminoacid.

One specific example of a synthesis according to Method K is as follows:

Method L

Two non-orthogonal protecting groups can be employed when both aminofunctionalities of the dibasic aminoacid are intended to befunctionalized with two moieties of the same additional aminoacid asexemplified by Method L. Deprotection in step d can unmask both aminogroups allowing functionalization of both of them at the same time (stepe).

One specific example of a synthesis according to Method L is as follows:

Methods of Use

In some embodiments, a method of treating or preventing a microbialinfection is provided, comprising administering to a subject sufferingfrom the microbial infection an amount effective to inhibit an effluxpump of said microbe of a compound described above. In some embodiments,the microbe is a bacteria and exhibits antibiotic resistance through anefflux pump mechanism. In some embodiments, the compound is administeredin conjuction with an antibiotic, wherein the compound is selected toreduce or eliminate tissue damage due to tissue accumulation of thecompound. In some embodiments, the subject is susceptible toaccumulating efflux pump inhibitors in tissue and an improvement overpreviously disclosed compounds is provided that includes selecting acompound that does not accumulate significantly in tissue.

In some embodiments, a method for treating or preventing growth ofantimicrobial-resistant microbes is provided, comprising contacting themicrobe with a compound of disclosed above and an antimicrobial agent.

In some embodiments, a method is provided for treating or preventing amicrobial infection, comprising identifying a subject that issusceptible to accumulation in tissue of a compound of Formula IIA:

and then administering to the subject a compound of formula II:

-   -   wherein:    -   D-AA-1 together with attached amine and carbonyl groups is a        first natural or artificial α-amino acid residue having an        (R)-configuration;    -   L-AA-1 together with attached amine and carbonyl groups is the        first α-amino acid residue but having an (S)-configuration;    -   D-AA-2 together with attached amine and carbonyl groups is a        second natural or artificial α-amino acid residue having an        (R)-configuration;    -   CG-1 is hydrogen or a carbon-linked capping group; and    -   CG-2 is a carbon-linked capping group, wherein when CG-1 is a        carbon-linked capping group, wherein CG-1 and CG-2 are        optionally linked together to form a 5- or 6-membered ring.

In some embodiments, a method if provided for treating a patient byadministering an efflux pump inhibitor in conjunction with anantibiotic, wherein the efflux pump inhibitor has the structure:

wherein AA-1 and AA-2 together with attached amine and carbonyl groupsrepresent a natural or artificial a-amino acid residue, and wherein themethod comprises ascertaining whether reduced cellular accumulation ofefflux pump inhibitor in the patient is desirable, and if so, selectingthe efflux pump inhibitor from those efflux pump inhibitors havingformula II:

-   -   wherein:    -   L-AA-1 is AA-2 having an (S)-configuration;    -   D-AA-2 is AA-2 having an (R)-configuration;    -   CG-1 is hydrogen or a carbon-linked capping group; and    -   CG-2 is a carbon-linked capping group, wherein when CG-1 is a        carbon-linked capping group, wherein CG-1 and CG-2 are        optionally linked together to form a 5- or 6-membered ring.

In some embodiments, a method is provided for treating a microbialinfection in an animal, specifically including in a mammal, by treatingan animal suffering from such an infection with an antimicrobial agentand an efflux pump inhibitor, which increase the susceptibility of themicrobe for that antimicrobial agent. Such efflux pump inhibitors can beselected from any of the compounds generically or specifically describedherein. In this way a microbe involved in the infection can be treatedusing the antimicrobial agent in smaller quantities, or can be treatedwith an antimicrobial agent, which is not therapeutically effective whenused in the absence of the efflux pump inhibitor. Thus, this method oftreatment is especially appropriate for the treatment of infectionsinvolving microbial strains that are difficult to treat using anantimicrobial agent alone due to a need for high dosage levels (whichcan cause undesirable side effects), or due to lack of any clinicallyeffective antimicrobial agents. However, it is also appropriate fortreating infections involving microbes that are susceptible toparticular antimicrobial agents as a way to reduce the dosage of thoseparticular agents. This can reduce the risk of side effects. It is alsoappropriate for treating infections involving microbes that aresusceptible to particular antimicrobial agents as a way of reducing thefrequency of selection of resistant microbes. In particular embodimentsthe microbe is a bacterium, which may, for example, be from any of thegroups or species indicated above.

In some embodiments, a method is provided for prophylactic treatment ofa mammal. In this method, an antimicrobial agent and an efflux pumpinhibitor is administered to a mammal at risk of a microbial infection,e.g., a bacterial infection. The efflux pump inhibitor can be selectedfrom any of the compounds generically or specifically described herein.

In some embodiments, a method is provided for enhancing theantimicrobial activity of an antimicrobial agent against a microbe, inwhich such a microbe is contacted with an efflux pump inhibitor, and anantibacterial agent. The efflux pump inhibitor can be selected from anyof the compounds generically or specifically described herein. Thus,this method makes an antimicrobial agent more effective against a cell,which expresses an efflux pump when the cell is treated with thecombination of an antimicrobial agent and an efflux pump inhibitor. Inparticular embodiments the microbe is a bacterium or a fungus, such asany of those indicated above; the antibacterial agent can be selectedfrom a number of structural classes of antibiotics including, e.g.,beta-lactams, glycopeptides, aminoglycosides, quinolones,oxazolidinones, tetracyclines, rifamycins, coumermycins, macrolides, andchloramphenicol. In particular embodiments an antibiotic of the aboveclasses can be as stated above.

In other embodiments, a method is provided for suppressing growth of amicrobe, e.g., a bacterium, expressing an efflux pump, e.g., anon-tetracycline-specific efflux pump. As illustrated by the case wherethe microbe is a bacterium, the method involves contacting thatbacterium with an efflux pump inhibitor, in the presence of aconcentration of antibacterial agent below the MIC of the bacterium. Theefflux pump inhibitor can be selected from any of the compoundsgenerically or specifically described herein. This method is useful, forexample, to prevent or cure contamination of a cell culture by abacterium possessing an efflux pump. However, it applies to anysituation where such growth suppression is desirable.

In some embodiments, any of the compounds generically or specificallydescribed herein may be administered as an efflux pump inhibitor eitheralone or, more preferably, in conjunction with another therapeuticagent. In some embodiments, any of the compounds generically orspecifically described herein may be administered as an efflux pumpinhibitor in conjunction with any of the antibacterial agentsspecifically or generically described herein, as well as with any otherantibacterial agent useful against the species of bacterium to betreated, when such bacteria do not utilize an efflux pump resistancemechanism. In some embodiments, the antibacterial agents areadministered at their usual recommended dosages. In other embodiments,the antibacterial agents are administered at reduced dosages, asdetermined by a physician. For all conventional antibacterials on themarket, and many in clinical development, dosage ranges and preferredroutes of administration are well established, and those dosages androutes can be used in conjunction with the efflux pump inhibitors of thepreferred embodiments. Reduced dosages of the antibacterials arecontemplated due to the increased efficacy of the antibacterial whencombined with an efflux pump inhibitor.

In some embodiments, a compound disclosed herein is administered alongwith an antimicrobial agent. The two agents may be administered in apredetermined ratio. For example, the agents may be administered in a1:1 ratio, 1:2 ratio, 2:1 ratio, etc. The agents may be administeredseparately, together, simultaneously, or sequentially. The agents may beadministered as a combined, fixed dosage form or as separate dosageforms.

In some embodiments, a subject is identified as infected with bacteriathat are resistant to an antimicrobial agent. The subject may then betreated with the antimicrobial agent in combination with a compounddisclosed herein. A subject may be identified as infected with bacteriathat are resistant based on observing an ineffective response of theinfection to the antimicrobial. Alternatively, the bacteria may becultured and identified as a known resistant strain by appropriatemicrobiological techniques known in the art.

In some embodiments, a subject is identified as a subject that isinfected with bacteria that are capable of developing resistance to anantimicrobial. The subject may then be treated with the antimicrobialagent in combination with a compound disclosed herein. A subject may beidentified as infected with bacteria that are capable of developingresistance by diagnosing the subject as having symptoms that arecharacteristic a bacterial infection with a bacteria species known tohave resistant strains or a with a bacteria that is a member of groupthat are known to have resistant strains. Alternatively, the bacteriamay be cultured and identified as a species known to have resistantstrains or a bacteria that is a member of group that are known to haveresistant strains.

In some embodiments, an efflux pump inhibitor is administered at a levelsufficient to overcome or suppress the emergence of efflux pump-mediatedresistance in bacteria. In some embodiments, this level produces aneffective efflux pump inhibitory concentration at the site of infection.In other embodiments, this level produces an effect equivalent toshutting down all efflux pumps in the bacteria.

In some embodiments, a subject is identified as a subject that is atrisk of infection with bacteria. The subject may then beprophylactically treated with an efflux pump inhibitor and anantimicrobial agent in order to prevent infection with a resistantbacterial strain. For example, subjects in environments likely to haveresistant bacteria, such as a hospital, may be prophylactically treated.

In some embodiments, a subject is treated with an efflux pump inhibitorthat is not otherwise generally effective as an antimicrobial. Thus, forexample, the MIC of the efflux pump inhibitor may be greater than about32 μg/ml, 64 μg/ml, 128 μg/ml, or 256 μg/ml.

Microbial Species

The microbial species to be inhibited through the use of efflux pumpinhibitors, such as the above-described soft drugs, can be from otherbacterial groups or species, such as one of the following: Pseudomonasaeruginosa, Pseudomonas fluorescens, Pseudomonas acidovorans,Pseudomonas alcaligenes, Pseudomonas putida, Stenotrophomonasmaltophilia, Burkholderia cepacia, Aeromonas hydrophilia, Escherichiacoli, Citrobacter freundii, Salmonella typhimurium, Salmonella typhi,Salmonella paratyphi, Salmonella enteritidis, Shigella dysenteriae,Shigella flexneri, Shigella sonnei, Enterobacter cloacae, Enterobacteraerogenes, Klebsiella pneumoniae, Klebsiella oxytoca, Serratiamarcescens, Francisella tularensis, Morganella morganii, Proteusmirabilis, Proteus vulgaris, Providencia alcalifaciens, Providenciarettgeri, Providencia stuartii, Acinetobacter calcoaceticus,Acinetobacter haemolyticus, Yersinia enterocolitica, Yersinia pestis,Yersinia pseudotuberculosis, Yersinia intermedia, Bordetella pertussis,Bordetella parapertussis, Bordetella bronchiseptica, Haemophilusinfluenzae, Haemophilus parainfluenzae, Haemophilus haemolyticus,Haemophilus parahaemolyticus, Haemophilus ducreyi, Pasteurellamultocida, Pasteurella haemolytica, Branhamella catarrhalis,Helicobacter pylori, Campylobacter fetus, Campylobacter jejuni,Campylobacter coli, Borrelia burgdorferi, Vibrio cholerae, Vibrioparahaemolyticus, Legionella pneumophila, Listeria monocytogenes,Neisseria gonorrhoeae, Neisseria meningitidis, Kingella, Moraxella,Gardnerella vaginalis, Bacteroides fragilis, Bacteroides distasonis,Bacteroides 3452A homology group, Bacteroides vulgatus, Bacteroidesovalus, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroideseggerthii, Bacteroides splanchnicus, Clostridium difficile,Mycobacterium tuberculosis, Mycobacterium avium, Mycobacteriumintracellulare, Mycobacterium leprae, Corynebacterium diphtheriae,Corynebacterium ulcerans, Streptococcus pneumoniae, Streptococcusagalactiae, Streptococcus pyogenes, Enterococcus faecalis, Enterococcusfaecium, Staphylococcus aureus, Staphylococcus epidermidis,Staphylococcus saprophyticus, Staphylococcus intermedius, Staphylococcushyicus subsp. hyicus, Staphylococcus haemolyticus, Staphylococcushominis, or Staphylococcus saccharolyticus.

A particularly appropriate example of a microbe appropriate for the useof an efflux pump inhibitor of the preferred embodiments is a pathogenicbacterial species, Pseudomonas aeruginosa, which is intrinsicallyresistant to many of the commonly used antibacterial agents. Exposingthis bacterium to an efflux pump inhibitor can significantly slow theexport of an antibacterial agent from the interior of the cell or theexport of siderophores. Therefore, if another antibacterial agent isadministered in conjunction with the efflux pump inhibitor of preferredembodiments, the antibacterial agent, which would otherwise bemaintained at a very low intracellular concentration by the exportprocess, can accumulate to a concentration, which will inhibit thegrowth of the bacterial cells. This growth inhibition can be due toeither bacteriostatic or bactericidal activity, depending on thespecific antibacterial agent used. While P. aeruginosa is an example ofan appropriate bacterium, other bacterial and microbial species maycontain similar broad substrate pumps, which actively export a varietyof antimicrobial agents, and thus can also be appropriate targets.

Antimicrobial Agents

In particular embodiments various antibacterial agents can be used incombination with the efflux pump inhibitors described herein. Theseinclude quinolones, tetracyclines, glycopeptides, aminoglycosides,β-lactams, rifamycins, macrolides/ketolides, oxazolidinones,coumermycins, and chloramphenicol. In particular embodiments, anantibiotic of the above classes can be, for example, one of thefollowing.

Beta-Lactam Antibiotics

Beta-lactam antibiotics include, but are not limited to, imipenem,meropenem, biapenem, cefaclor, cefadroxil, cefamandole, cefatrizine,cefazedone, cefazolin, cefixime, cefmenoxime, cefodizime, cefonicid,cefoperazone, ceforanide, cefotaxime, cefotiam, cefpimizole,cefpiramide, cefpodoxime, cefsulodin, ceftazidime, cefteram, ceftezole,ceftibuten, ceftizoxime, ceftriaxone, cefuroxime, cefuzonam,cephaacetrile, cephalexin, cephaloglycin, cephaloridine, cephalothin,cephapirin, cephradine, cefinetazole, cefoxitin, cefotetan, azthreonam,carumonam, flomoxef, moxalactam, amidinocillin, amoxicillin, ampicillin,azlocillin, carbenicillin, benzylpenicillin, carfecillin, cloxacillin,dicloxacillin, methicillin, mezlocillin, nafcillin, oxacillin,penicillin G, piperacillin, sulbenicillin, temocillin, ticarcillin,cefditoren, SC004, KY-020, cefdinir, ceftibuten, FK-312, S-1090,CP-0467, BK-218, FK-037, DQ-2556, FK-518, cefozopran, ME1228, KP-736,CP-6232, Ro 09-1227, OPC-20000, and LY206763.

Macrolides

Macrolides include, but are not limited to, azithromycin,clarithromycin, erythromycin, oleandomycin, rokitamycin, rosaramicin,roxithromycin, and troleandomycin.

Ketolides

Ketolides include, but are not limited to, telithromycin andcethrimycin.

Quinolones

Quinolones include, but are not limited to, amifloxacin, cinoxacin,ciprofloxacin, enoxacin, fleroxacin, flumequine, lomefloxacin, nalidixicacid, norfloxacin, ofloxacin, levofloxacin, oxolinic acid, pefloxacin,rosoxacin, temafloxacin, tosufloxacin, sparfloxacin, clinafloxacin,moxifloxacin; gemifloxacin; garenofloxacin; PD131628, PD138312,PD140248, Q-35, AM-1155, NM394, T-3761, rufloxacin, OPC-17116, DU-6859a(see, e.g., Sato, K. et al., 1992, Antimicrob Agents Chemother.37:1491-98), and DV-7751a (see, e.g., Tanaka, M. et al., 1992,Antimicrob. Agents Chemother. 37:2212-18).

Tetracyclines, Glycylcyclines and Oxazolidinones

Tetracyclines, glycylcyclines, and oxazolidinones include, but are notlimited to, chlortetracycline, demeclocycline, doxycycline, lymecycline,methacycline, minocycline, oxytetracycline, tetracycline, tigecycline,linezolide, and eperozolid.

Aminoglycosides

Aminoglycosides include, but are not limited to amikacin, arbekacin,butirosin, dibekacin, fortimicins, gentamicin, kanamycin, meomycin,netilmicin, ribostamycin, sisomicin, spectinomycin, streptomycin, andtobramycin.

Lincosamides

Lincosamides include, but are not limited to, clindamycin andlincomycin.

Screening for Efflux Pump Inhibitors

Potential efflux pump inhibitor compounds can be tested for theirability to inhibit multi-drug resistance efflux pumps of variousmicrobes and to potentiate various antimicrobial agents by using themethods described herein as well as those known in the art. For example,strains of microbes known to overexpress efflux pumps may be treatedwith the antimicrobial agent with and without the test efflux pumpinhibitor compound. A checkerboard assay may be used with varyingconcentrations of both antimicrobial agent and test compound todetermine the relative concentrations at which potentiation is observed.

In one non-limiting example, treatment of P. aeruginosa with a testcompound allows obtaining one or more of the following biologicaleffects:

-   -   pa-301) P. aeruginosa strains will become susceptible to        antibiotics that could not be used for treatment of pseudomonas        infections, or become more susceptible to antibiotics, become        more susceptible to antibiotics currently used for treatment of        pseudomonas infections.    -   2) P. aeruginosa strains which developed resistance to        antibiotics currently used for treatment of pseudomonas        infections will become susceptible to these antibiotics.    -   3) Inhibition of the pump will result in a decreased frequency        of resistance development to antibiotic, which is a substrate of        the pump.

Obtaining even one of these effects provides a potential therapeutictreatment for infections by this bacterium. Also, similar pumps arefound in other microorganisms. Some or all of the above effects can alsobe obtained with those microbes, and they are therefore also appropriatetargets for detecting or using efflux pump inhibitors.

Pharmaceutical Compositions

In another aspect, the present disclosure relates to a pharmaceuticalcomposition comprising a physiologically acceptable surface activeagents, carriers, diluents, excipients, smoothing agents, suspensionagents, film forming substances, and coating assistants, or acombination thereof; and a compound disclosed herein. Acceptablecarriers or diluents for therapeutic use are well known in thepharmaceutical art, and are described, for example, in Remington'sPharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, Pa.(1990), which is incorporated herein by reference in its entirety.Preservatives, stabilizers, dyes, sweeteners, fragrances, flavoringagents, and the like may be provided in the pharmaceutical composition.For example, sodium benzoate, ascorbic acid and esters ofp-hydroxybenzoic acid may be added as preservatives. In addition,antioxidants and suspending agents may be used. In various embodiments,alcohols, esters, sulfated aliphatic alcohols, and the like may be usedas surface active agents; sucrose, glucose, lactose, starch,crystallized cellulose, mannitol, light anhydrous silicate, magnesiumaluminate, magnesium methasilicate aluminate, synthetic aluminumsilicate, calcium carbonate, sodium acid carbonate, calcium hydrogenphosphate, calcium carboxymethyl cellulose, and the like may be used asexcipients; magnesium stearate, talc, hardened oil and the like may beused as smoothing agents; coconut oil, olive oil, sesame oil, peanutoil, soya may be used as suspension agents or lubricants; celluloseacetate phthalate as a derivative of a carbohydrate such as cellulose orsugar, or methylacetate-methacrylate copolymer as a derivative ofpolyvinyl may be used as suspension agents; and plasticizers such asester phthalates and the like may be used as suspension agents.

The term “pharmaceutical composition” refers to a mixture of a compounddisclosed herein with other chemical components, such as diluents orcarriers. The pharmaceutical composition facilitates administration ofthe compound to an organism. Multiple techniques of administering acompound exist in the art including, but not limited to, oral,injection, aerosol, parenteral, and topical administration.Pharmaceutical compositions can also be obtained by reacting compoundswith inorganic or organic acids such as hydrochloric acid, hydrobromicacid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid,ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and thelike.

The term “carrier” defines a chemical compound that facilitates theincorporation of a compound into cells or tissues. For example dimethylsulfoxide (DMSO) is a commonly utilized carrier as it facilitates theuptake of many organic compounds into the cells or tissues of anorganism.

The term “diluent” defines chemical compounds diluted in water that willdissolve the compound of interest as well as stabilize the biologicallyactive form of the compound. Salts dissolved in buffered solutions areutilized as diluents in the art. One commonly used buffered solution isphosphate buffered saline because it mimics the salt conditions of humanblood. Since buffer salts can control the pH of a solution at lowconcentrations, a buffered diluent rarely modifies the biologicalactivity of a compound.

The term “physiologically acceptable” defines a carrier or diluent thatdoes not abrogate the biological activity and properties of thecompound.

The pharmaceutical compositions described herein can be administered toa human patient per se, or in pharmaceutical compositions where they aremixed with other active ingredients, as in combination therapy, orsuitable carriers or excipient(s). Techniques for formulation andadministration of the compounds of the instant application may be foundin “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton,Pa., 18th edition, 1990.

Suitable routes of administration may, for example, include oral,rectal, transmucosal, topical, or intestinal administration; parenteraldelivery, including intramuscular, subcutaneous, intravenous,intramedullary injections, as well as intrathecal, directintraventricular, intraperitoneal, intranasal, or intraocularinjections. The compounds can also be administered in sustained orcontrolled release dosage forms, including depot injections, osmoticpumps, pills, transdermal (including electrotransport) patches, and thelike, for prolonged and/or timed, pulsed administration at apredetermined rate.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is itself known, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or tabletting processes.

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen. Any of the well-knowntechniques, carriers, and excipients may be used as suitable and asunderstood in the art; e.g., in Remington's Pharmaceutical Sciences,above.

Injectables can be prepared in conventional forms, either as liquidsolutions or suspensions, solid forms suitable for solution orsuspension in liquid prior to injection, or as emulsions. Suitableexcipients are, for example, water, saline, dextrose, mannitol, lactose,lecithin, albumin, sodium glutamate, cysteine hydrochloride, and thelike. In addition, if desired, the injectable pharmaceuticalcompositions may contain minor amounts of nontoxic auxiliary substances,such as wetting agents, pH buffering agents, and the like.Physiologically compatible buffers include, but are not limited to,Hanks's solution, Ringer's solution, or physiological saline buffer. Ifdesired, absorption enhancing preparations (for example, liposomes), maybe utilized.

For transmucosal administration, penetrants appropriate to the barrierto be permeated may be used in the formulation.

Pharmaceutical formulations for parenteral administration, e.g., bybolus injection or continuous infusion, include aqueous solutions of theactive compounds in water-soluble form. Additionally, suspensions of theactive compounds may be prepared as appropriate oily injectionsuspensions. Suitable lipophilic solvents or vehicles include fatty oilssuch as sesame oil, or other organic oils such as soybean, grapefruit oralmond oils, or synthetic fatty acid esters, such as ethyl oleate ortriglycerides, or liposomes. Aqueous injection suspensions may containsubstances which increase the viscosity of the suspension, such assodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, thesuspension may also contain suitable stabilizers or agents that increasethe solubility of the compounds to allow for the preparation of highlyconcentrated solutions. Formulations for injection may be presented inunit dosage form, e.g., in ampoules or in multi-dose containers, with anadded preservative. The compositions may take such forms as suspensions,solutions or emulsions in oily or aqueous vehicles, and may containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

For oral administration, the compounds can be formulated readily bycombining the active compounds with pharmaceutically acceptable carrierswell known in the art. Such carriers enable the compounds of theinvention to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions and the like, for oralingestion by a patient to be treated. Pharmaceutical preparations fororal use can be obtained by combining the active compounds with solidexcipient, optionally grinding a resulting mixture, and processing themixture of granules, after adding suitable auxiliaries, if desired, toobtain tablets or dragee cores. Suitable excipients are, in particular,fillers such as sugars, including lactose, sucrose, mannitol, orsorbitol; cellulose preparations such as, for example, maize starch,wheat starch, rice starch, potato starch, gelatin, gum tragacanth,methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired,disintegrating agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodiumalginate. Dragee cores are provided with suitable coatings. For thispurpose, concentrated sugar solutions may be used, which may optionallycontain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel,polyethylene glycol, and/or titanium dioxide, lacquer solutions, andsuitable organic solvents or solvent mixtures. Dyestuffs or pigments maybe added to the tablets or dragee coatings for identification or tocharacterize different combinations of active compound doses. For thispurpose, concentrated sugar solutions may be used, which may optionallycontain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel,polyethylene glycol, and/or titanium dioxide, lacquer solutions, andsuitable organic solvents or solvent mixtures. Dyestuffs or pigments maybe added to the tablets or dragee coatings for identification or tocharacterize different combinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. All formulations fororal administration should be in dosages suitable for suchadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebulizer, with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of, e.g., gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

Further disclosed herein are various pharmaceutical compositions wellknown in the pharmaceutical art for uses that include intraocular,intranasal, and intraauricular delivery. Suitable penetrants for theseuses are generally known in the art. Pharmaceutical compositions forintraocular delivery include aqueous ophthalmic solutions of the activecompounds in water-soluble form, such as eyedrops, or in gellan gum(Shedden et al., Clin. Ther., 23(3):440-50 (2001)) or hydrogels (Mayeret al., Ophthalmologica, 210(2):101-3 (1996)); ophthalmic ointments;ophthalmic suspensions, such as microparticulates, drug-containing smallpolymeric particles that are suspended in a liquid carrier medium(Joshi, A., J. Ocul. Pharmacol., 10(1):29-45 (1994)), lipid-solubleformulations (Alm et al., Prog. Clin. Biol. Res., 312:447-58 (1989)),and microspheres (Mordenti, Toxicol. Sci., 52(1):101-6 (1999)); andocular inserts. All of the above-mentioned references, are incorporatedherein by reference in their entireties. Such suitable pharmaceuticalformulations are most often and preferably formulated to be sterile,isotonic and buffered for stability and comfort. Pharmaceuticalcompositions for intranasal delviery may also include drops and spraysoften prepared to simulate in many respects nasal secretions to ensuremaintenance of normal ciliary action. As disclosed in Remington'sPharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, Pa.(1990), which is incorporated herein by reference in its entirety, andwell-known to those skilled in the art, suitable formulations are mostoften and preferably isotonic, slightly buffered to maintain a pH of 5.5to 6.5, and most often and preferably include antimicrobialpreservatives and appropriate drug stabilizers. Pharmaceuticalformulations for intraauricular delivery include suspensions andointments for topical application in the ear. Common solvents for suchaural formulations include glycerin and water.

The compounds may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

For hydrophobic compounds, a suitable pharmaceutical carrier may be acosolvent system comprising benzyl alcohol, a nonpolar surfactant, awater-miscible organic polymer, and an aqueous phase. A common cosolventsystem used is the VPD co-solvent system, which is a solution of 3% w/vbenzyl alcohol, 8% w/v of the nonpolar surfactant Polysorbate 80™, and65% w/v polyethylene glycol 300, made up to volume in absolute ethanol.Naturally, the proportions of a co-solvent system may be variedconsiderably without destroying its solubility and toxicitycharacteristics. Furthermore, the identity of the co-solvent componentsmay be varied: for example, other low-toxicity nonpolar surfactants maybe used instead of POLYSORBATE 80™; the fraction size of polyethyleneglycol may be varied; other biocompatible polymers may replacepolyethylene glycol, e.g., polyvinyl pyrrolidone; and other sugars orpolysaccharides may substitute for dextrose.

Alternatively, other delivery systems for hydrophobic pharmaceuticalcompounds may be employed. Liposomes and emulsions are well knownexamples of delivery vehicles or carriers for hydrophobic drugs. Certainorganic solvents such as dimethylsulfoxide also may be employed,although usually at the cost of greater toxicity. Additionally, thecompounds may be delivered using a sustained-release system, such assemipermeable matrices of solid hydrophobic polymers containing thetherapeutic agent. Various sustained-release materials have beenestablished and are well known by those skilled in the art.Sustained-release capsules may, depending on their chemical nature,release the compounds for a few weeks up to over 100 days. Depending onthe chemical nature and the biological stability of the therapeuticreagent, additional strategies for protein stabilization may beemployed.

Agents intended to be administered intracellularly may be administeredusing techniques well known to those of ordinary skill in the art. Forexample, such agents may be encapsulated into liposomes. All moleculespresent in an aqueous solution at the time of liposome formation areincorporated into the aqueous interior. The liposomal contents are bothprotected from the external micro-environment and, because liposomesfuse with cell membranes, are efficiently delivered into the cellcytoplasm. The liposome may be coated with a tissue-specific antibody.The liposomes will be targeted to and taken up selectively by thedesired organ. Alternatively, small hydrophobic organic molecules may bedirectly administered intracellularly.

Additional therapeutic or diagnostic agents may be incorporated into thepharmaceutical compositions. Alternatively or additionally,pharmaceutical compositions may be combined with other compositions thatcontain other therapeutic or diagnostic agents.

Methods of Administration

The compounds or pharmaceutical compositions may be administered to thepatient by any suitable means. Non-limiting examples of methods ofadministration include, among others, (a) administration though oralpathways, which administration includes administration in capsule,tablet, granule, spray, syrup, or other such forms; (b) administrationthrough non-oral pathways such as rectal, vaginal, intraurethral,intraocular, intranasal, or intraauricular, which administrationincludes administration as an aqueous suspension, an oily preparation orthe like or as a drip, spray, suppository, salve, ointment or the like;(c) administration via injection, subcutaneously, intraperitoneally,intravenously, intramuscularly, intradermally, intraorbitally,intracapsularly, intraspinally, intrasternally, or the like, includinginfusion pump delivery; (d) administration locally such as by injectiondirectly in the renal or cardiac area, e.g., by depot implantation; aswell as (e) administration topically; as deemed appropriate by those ofskill in the art for bringing the compound of the invention into contactwith living tissue.

Pharmaceutical compositions suitable for administration includecompositions where the active ingredients are contained in an amounteffective to achieve its intended purpose. The therapeutically effectiveamount of the compounds disclosed herein required as a dose will dependon the route of administration, the type of animal, including human,being treated, and the physical characteristics of the specific animalunder consideration. The dose can be tailored to achieve a desiredeffect, but will depend on such factors as weight, diet, concurrentmedication and other factors which those skilled in the medical artswill recognize. More specifically, a therapeutically effective amountmeans an amount of compound effective to prevent, alleviate orameliorate symptoms of disease or prolong the survival of the subjectbeing treated. Determination of a therapeutically effective amount iswell within the capability of those skilled in the art, especially inlight of the detailed disclosure provided herein.

As will be readily apparent to one skilled in the art, the useful invivo dosage to be administered and the particular mode of administrationwill vary depending upon the age, weight and mammalian species treated,the particular compounds employed, and the specific use for which thesecompounds are employed. The determination of effective dosage levels,that is the dosage levels necessary to achieve the desired result, canbe accomplished by one skilled in the art using routine pharmacologicalmethods. Typically, human clinical applications of products arecommenced at lower dosage levels, with dosage level being increaseduntil the desired effect is achieved. Alternatively, acceptable in vitrostudies can be used to establish useful doses and routes ofadministration of the compositions identified by the present methodsusing established pharmacological methods.

In non-human animal studies, applications of potential products arecommenced at higher dosage levels, with dosage being decreased until thedesired effect is no longer achieved or adverse side effects disappear.The dosage may range broadly, depending upon the desired effects and thetherapeutic indication. Typically, dosages may be between about 10microgram/kg and 100 mg/kg body weight, preferably between about 100microgram/kg and 10 mg/kg body weight. Alternatively dosages may bebased and calculated upon the surface area of the patient, as understoodby those of skill in the art.

The exact formulation, route of administration and dosage for thepharmaceutical compositions of the present invention can be chosen bythe individual physician in view of the patient's condition. (See e.g.,Fingl et al. 1975, in “The Pharmacological Basis of Therapeutics”, whichis hereby incorporated herein by reference in its entirety, withparticular reference to Ch. 1, p. 1). Typically, the dose range of thecomposition administered to the patient can be from about 0.5 to 1000mg/kg of the patient's body weight. The dosage may be a single one or aseries of two or more given in the course of one or more days, as isneeded by the patient. In instances where human dosages for compoundshave been established for at least some condition, the present inventionwill use those same dosages, or dosages that are between about 0.1% and500%, more preferably between about 25% and 250% of the establishedhuman dosage. Where no human dosage is established, as will be the casefor newly-discovered pharmaceutical compounds, a suitable human dosagecan be inferred from ED₅₀ or ID₅₀ values, or other appropriate valuesderived from in vitro or in vivo studies, as qualified by toxicitystudies and efficacy studies in animals.

It should be noted that the attending physician would know how to andwhen to terminate, interrupt, or adjust administration due to toxicityor organ dysfunctions. Conversely, the attending physician would alsoknow to adjust treatment to higher levels if the clinical response werenot adequate (precluding toxicity). The magnitude of an administrateddose in the management of the disorder of interest will vary with theseverity of the condition to be treated and to the route ofadministration. The severity of the condition may, for example, beevaluated, in part, by standard prognostic evaluation methods. Further,the dose and perhaps dose frequency, will also vary according to theage, body weight, and response of the individual patient. A programcomparable to that discussed above may be used in veterinary medicine.

Although the exact dosage will be determined on a drug-by-drug basis, inmost cases, some generalizations regarding the dosage can be made. Thedaily dosage regimen for an adult human patient may be, for example, anoral dose of between 0.1 mg and 2000 mg of each active ingredient,preferably between 1 mg and 500 mg, e.g. 5 to 200 mg. In otherembodiments, an intravenous, subcutaneous, or intramuscular dose of eachactive ingredient of between 0.01 mg and 100 mg, preferably between 0.1mg and 60 mg, e.g. 1 to 40 mg is used. In cases of administration of apharmaceutically acceptable salt, dosages may be calculated as the freebase. In some embodiments, the composition is administered 1 to 4 timesper day. Alternatively the compositions of the invention may beadministered by continuous intravenous infusion, preferably at a dose ofeach active ingredient up to 1000 mg per day. As will be understood bythose of skill in the art, in certain situations it may be necessary toadminister the compounds disclosed herein in amounts that exceed, oreven far exceed, the above-stated, preferred dosage range in order toeffectively and aggressively treat particularly aggressive diseases orinfections. In some embodiments, the compounds will be administered fora period of continuous therapy, for example for a week or more, or formonths or years.

Dosage amount and interval may be adjusted individually to provideplasma levels of the active moiety which are sufficient to maintain themodulating effects, or minimal effective concentration (MEC). The MECwill vary for each compound but can be estimated from in vitro data.Dosages necessary to achieve the MEC will depend on individualcharacteristics and route of administration. However, HPLC assays orbioassays can be used to determine plasma concentrations.

Dosage intervals can also be determined using MEC value. Compositionsshould be administered using a regimen which maintains plasma levelsabove the MEC for 10-90% of the time, preferably between 30-90% and mostpreferably between 50-90%.

In cases of local administration or selective uptake, the effectivelocal concentration of the drug may not be related to plasmaconcentration.

The amount of composition administered may be dependent on the subjectbeing treated, on the subject's weight, the severity of the affliction,the manner of administration and the judgment of the prescribingphysician.

Compounds disclosed herein can be evaluated for efficacy and toxicityusing known methods. For example, the toxicology of a particularcompound, or of a subset of the compounds, sharing certain chemicalmoieties, may be established by determining in vitro toxicity towards acell line, such as a mammalian, and preferably human, cell line. Theresults of such studies are often predictive of toxicity in animals,such as mammals, or more specifically, humans. Alternatively, thetoxicity of particular compounds in an animal model, such as mice, rats,rabbits, or monkeys, may be determined using known methods. The efficacyof a particular compound may be established using several recognizedmethods, such as in vitro methods, animal models, or human clinicaltrials. Recognized in vitro models exist for nearly every class ofcondition, including but not limited to cancer, cardiovascular disease,and various immune dysfunction. Similarly, acceptable animal models maybe used to establish efficacy of chemicals to treat such conditions.When selecting a model to determine efficacy, the skilled artisan can beguided by the state of the art to choose an appropriate model, dose, androute of administration, and regime. Of course, human clinical trialscan also be used to determine the efficacy of a compound in humans.

The compositions may, if desired, be presented in a pack or dispenserdevice which may contain one or more unit dosage forms containing theactive ingredient. The pack may for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device may beaccompanied by instructions for administration. The pack or dispensermay also be accompanied with a notice associated with the container inform prescribed by a governmental agency regulating the manufacture,use, or sale of pharmaceuticals, which notice is reflective of approvalby the agency of the form of the drug for human or veterinaryadministration. Such notice, for example, may be the labeling approvedby the U.S. Food and Drug Administration for prescription drugs, or theapproved product insert. Compositions comprising a compound of theinvention formulated in a compatible pharmaceutical carrier may also beprepared, placed in an appropriate container, and labeled for treatmentof an indicated condition.

EXAMPLES

The following examples serve to more fully describe the preferredembodiments, as well as to set forth the best modes contemplated forcarrying out various aspects of the preferred embodiments. It isunderstood that these examples in no way serve to limit the true scopeof this invention, but rather are presented for illustrative purposes.All references cited herein are incorporated by reference in theirentirety.

Synthesis of Compounds

The following synthetic methods were used to prepare all compoundsdescribed as examples below. Unless otherwise stated all compounds weremade in the form of mono methanesulfonate salts and the NMR spectra arethat of methanesulfonates of the title compounds. In several cases, thestep of converting initially obtained trifluoroacetate salt intomethanesulfonate was omitted and the compounds were characterized andtested in trifluoroacetate form. All NMR spectra were recorded inDMSO-d₆. The methanesulfonic acid peak 2.34 ppm (3H) was omitted formNMR spectra of methanesulfonates listed below. Unless otherwiseindicated, mass spectra were obtained using the electrospray ionizationmethod (MS-ESI). When indicated as MS-EI, electron impact ionization wasused.

Example 1 Method F: 2-(S),5-Diamino-pentanoic acid[3-phenyl-1-(R)-(4-phenyl-thiazol-2-ylcarbamoyl)-propyl]-amidemono-methanesulfonate (Compound 59) (A) D-Homophenylalanine Benzyl esterTosylate

A solution of D-Homophenylalanine hydrochloride (1.7 g, 7.93 mmol),benzyl alcohol (7.2 mL, 64.0 mmol) and p-toluenesulfonic acidmonohydrate (1.8 g, 9.5 mmol) in benzene (30 mL) was heated at reflux inDean Stark apparatus during 5 hrs, after which time additional benzene(100 mL) was distilled from reaction mixture. The residue was trituratedwith diethyl ether, the solid was filtered and dried to give titleproduct (2.93 g).

¹H NMR (DMSO-d₆) 2.00-2.08 (m, 2H),2.28 (s, 3H), 2.50-2.57 (m, 1H),2.66-2.74 (m, 1H), 5.25 (dd, J=17 Hz, J=12 Hz, 2H), 7.10-7.49 (m, 9H),8.42 (brs, 3H)

(B) N_(a)N_(d)-bis-Boc-L-Ornithyl-D-Homophenylalanine Benzyl ester

D-Homophenylalanine Benzyl ester Tosylate (2.48 g, 5.6 mmol) wassuspended in ethyl acetate (25 mL) and a saturated solution of sodiumbicarbonate in water was added in portions (25 mL) with stirring at 25°C. The water layer was extracted with ethyl acetate (3×10 mL). Thecombined organic extract was dried over anhydrous sodium sulfate,filtered and concentrated to dryness to afford D-HomophenylalanineBenzyl ester. The ester was dissolved in acetonitile (25 mL).N_(a)N_(d)-bis-Boc-L-Ornithine (2.0 g, 6.0 mmol) was then added followedby N-hydroxysuccinimide (50 mg) and dicyclohexylcarbodiimide (1.28 g,6.2 mmol). The reaction was stirred at 25° C. for 30 hr, filtered andthe filtrate concentrated to dryness to afford crude product. It waspurified by column chromatography on silica gel (hexane:ethylacetate=3:1), which gave the title product (2.80 g) as white solid.

¹H NMR (DMSO-d₆) 1.35 (s, 9H), 1.38 (s, 9H), 1.38-1.74 (m, 4H),1.87-2.00 (m, 2H), 2.50-2.60 (m, 2H), 2.88 (q, J=6 Hz, 2H), 3.95-4.00(m, 1H), 4.19-4.24 (m, 1H), 5.25 (dd, J=17 Hz, J=12 Hz, 2H), 6.75-6.80(m, 2H), 7.24-7.38 (m, 10H), 8.31 (d, J=8 Hz, 1H)

MS (EI) found for C₃₂H₄₃N₃O₇ M⁺=583

(C) N_(a)N_(d)-bis-Boc-L-Ornithyl-D-Homophenylalanine

A solution of N_(a)N_(d)-bis-Boc-L-Ornithyl-D-Homophenylalanine Benzylester (2.80 g, 4.80 mmol) in methanol (200 mL) was treated with 10% Pd/Ccatalyst (0:2 g) and stirred in the atmosphere of hydrogen at 25° C. for3 hr. The catalyst was removed by filtration through Celite pad and thefiltrate evaporated to dryness to give the title product (2.48 g).

¹H NMR (DMSO-d₆) 1.35 (s, 9H), 1.38 (s, 9H), 1.38-1.55 (m, 4H),1.82-1.92 (m, 1H), 1.93-2.01 (m, 1H), 2.50-2.63 (m, 2H), 2.90 (q, J=6Hz, 2H), 3.95-4.00 (m, 1H), 4.10-4.16 (m, 1H), 6.77-6.79 (m, 2H),7.15-7.29 (m, 5H), 8.09 (d, J=8 Hz, 1H)

(D) N_(a)N_(d)-bis-Boc-L-Ornithyl-D-Homophenylalanine(2-amino-4-phenylthiazole) amide

A solution of N_(a)N_(d)-bis-Boc-L-Ornithyl-D-Homophenylalanine (0.3 g,0.6 mmol) in methylene chloride (10 mL) was treated with2-amino-4-phenylthiazole (0.16 g, 0.9 mmol) followed byN-hydroxysuccinimide (10 mg) and dicyclohexylcarbodiimide (0.15 g, 0.7mmol). The reaction was stirred at 25° C. for 18 hr (HPLC monitoringRP-18, 250×4 mm, water:acetonitrile:triethylamine:aceticacid=500:500:0.6:1 (mL), UV detector, λ=254 nm), filtered and thefiltrate washed with 1M hydrochloric acid (25 mL), saturated solution ofsodium bicarbonate (25 mL) and water (25 mL). The organic layer wasdried over anhydrous sodium sulfate, filtered and concentrated todryness to afford crude product. The product was purified by columnchromatography on silica gel (hexane:ethyl acetate=1:1), which gave thetitle product (0.37 g) as white solid.

¹H NMR (DMSO-d₆) 1.35 (s, 9H), 1.38 (s, 9H), 1.42-1.52 (m, 2H),1.60-1.64 (m, 1H), 1.70-1.74 (m, 1H), 1.90-2.00 (m, 1H), 2.08-2.17 (m,1H), 2.51-2.60 (m, 1H), 2.63-2.68 (m, 1H), 2.93-2.94 (m, 2H), 4.00 (brs,1H), 4.53 (brs, 1H), 6.76-6.79 (m, 1H), 6.89-6.95 (m, 1H), 7.16-7.34 (m,8H), 7.39-7.43 (m, 2H), 7.62 (m, 1H), 7.88-7.91 (m, 2H); MS (EI) foundfor C₃₄H₄₅N₅O₆S (EI) M⁺=651

(E) L-Ornithyl-D-Homophenylalanine (2-amino-4-phenylthiazole) amideMesylate

N_(a)N_(d)-bis-Boc-L-Ornithyl-D-Homophenylalanine(2-amino-4-phenylthiazole) amide was converted into mono-mesylate saltof the title compound according to the procedure described below indetail for compound 3.

Example 2 Method Ha: 2-(S),5-Diamino-pentanoic acid[3-phenyl-1-(R)-(quinolin-3-ylcarbamoyl)-propyl]-amidemono-methanesulfonate (compound 3) (A) N-Boc-D-HomophenylalanineQuinoline-3-amide

A solution of N-Boc-D-Homophenylalanine (3.0 g, 10.7 mmol) in ethylacetate (100 mL) was treated with 3-aminoquinoline (3.08 g, 21.4 mmol)followed by dicyclohexylcarbodiimide (2.31 g, 11.2 mmol). The reactionwas stirred at 25° C. for 3 hr (HPLC monitoring RP-18, 250×4 mm,water:acetonitrile:triethylamine:acetic acid=500:500:0.6:1 (mL), UVdetector, λ=254 nm), filtered and the filtrate washed with 1Mhydrochloric acid (25 mL), saturated solution of sodium bicarbonate (25mL) and water (25 mL). The organic layer was dried over anhydrous sodiumsulfate, filtered and concentrated to dryness to afford title compound(4.3 g) as white solid.

¹H NMR (DMSO-d₆) 1.40 (s, 9H), 1.90-2.05 (m, 2H), 2.58-2.78 (m, 2H),4.15-4.17 (m, 1H), 7.15-7.33 (m, 6H), 7.54-7.58 (m, 1H), 7.61-7.66 (m,1H), 7.93 (t, J=9 Hz, 2H), 8.69 (d, J=2 Hz, 1H), 8.92 (d, J=2 Hz, 1H),10.47 (s, 1H)

MS found for C₂₄H₂₇N₃O₃ (M+H)⁺=406

(B) N_(a)N_(d)-bis-Boc-L-Ornithyl-D-Homophenylalanine Quinoline-3-amide

A solution of N_(a)N_(d)-bis-Boc-L-Ornithine (2.79 g, 8.4 mmol),triethylamine (1.29 mL, 9.2 mmol) and methylene chloride (90 mL) wasstirred at 25° C. for 10 min, cooled to −10° C. and treated with ethylchloroformate (0.80 mL, 8.4 mmol). The mixture was stirred at −10° C.for 2 hrs. During this time N-Boc-D-Homophenylalanine Quinoline-3-amide(2.27 g, 5.6 mmol) was treated with trifluoroacetic acid (15 mL) at 25°C. for 1 hr. The solution was concentrated to dryness and treated withdiethyl ether (10 mL). The solid was filtered and dried. It wassuspended in methylene chloride (40 mL) and neutralized withtriethylamine (1.6 mL, 11.2 mmol). The resultant solution was addeddropwise to the mixed anhydride and the mixture was allowed to warm upto 25° C. and stirred for an additional 1 hr. Saturated sodiumbicarbonate (30 mL) was added, the organic layer was washed with water(30 mL), dried over anhydrous sodium sulfate, filtered and concentratedto dryness to afford crude product. It was purified by columnchromatography on silica gel (ethyl acetate:hexane=2:1) to give thetitle compound (2.55 g) as white solid.

¹H NMR (DMSO-d₆) 1.35 (s, 9H), 1.36 (s, 9H), 1.40-1.66 (m, 4H) 1.91-1.99(m, 1H), 2.10-2.18 (m, 1H), 2.56-2.76 (m, 2H), 2.92 (q, J=6 Hz, 2H),3.97-4.0 (m, 1H) 4.44-4.49 (m, 1H), 6.78 (t, J=5 Hz, 1H), 7.03 (d, J=7Hz, 1H), 7.15-7.29 (m, 5H), 7.54-7.58 (m, 1H), 7.62-7.66 (m, 1H),7.88-7.95 (m, 2H), 8.44 (d, J=8 Hz, 1H), 8.69 (d, J=2 Hz, 1H), 8.97 (d,J=2 Hz, 1H), 10.24 (s, 1H)

MS found for C₃₄H₄₅N₅O₆ (M+H)⁺=620

(C) L-Ornithyl-D-Homophenylalanine Quinoline-3-amide Mesylate

N_(a)N_(d)-bis-Boc-L-Ornithyl-D-Homophenylalanine Quinoline-3-amide(3.26 g, 5.3 mmol) was treated with trifluoroacetic acid (25 ml) at 25°C. After 1 hr the reaction was concentrated in vacuo. The residue wasdissolved in water (10 mL) and loaded on HP 20 filled column. It waswashed with 1.5% sodium bicarbonate solution (600 mL), then water (500mL), then eluted with water:methanol 2:8 vol (600 mL). Fractionscontaining organic material (TLC monitoring, ethanol:ammonia=8:2 elutionon silica gel plates) were concentrated in vacuo and dried to affordfree amine of the title compound. It was suspended in water (50 mL) andmethanesulfonic acid (0.32 mL, 5 mmol) was added. Slightly turbulentsolution was filtered through Celite pad and the filtrate concentratedto dryness. The residue was triturated with diethyl ether (50 mL), thesolid was filtered and dried to give title compound (1.48 g).

¹H NMR (DMSO-d₆) 1.48-1.57 (m, 1H), 1.62-1.77 (m, 2H), 1.94-2.04 (m,1H), 2.07-2.16 (m, 1H), 2.60-2.67 (m, 1H), 2.70-2.77 (m, 1H), 2.78-2.84(m, 2H), 3.39 (brs, 1H), 4.55 (brs, 1H), 6.03 (brs, 4H), 7.16-7.30 (m,5H), 7.55-7.60 (m, 1H), 7.63-7.67 (m, 1H), 7.94 (dd, J=14 Hz, J=8 Hz,2H), 8.50 (brs, 1H), 8.68 (d, J=2 Hz 1H), 8.95 (d, J=2 Hz, 1H), 10.61(s, 1H)

MS found for C₂₄H₂₉N₅O₂ (M+H)⁺=420

Example 3 Method Hb: 4-(S)-Aminomethyl-pyrrolidine-2-(S)-carboxylic acid[3-phenyl-1-(R)-(quinolin-3-ylcarbamoyl)-propyl]-amidemono-methanesulfonate (compound 15) (A) D-HomophenylalanineQuinoline-3-amide

N-Boc-D-homophenylalanine Quinoline-3-amide (2.6 g, 6.42 mmol, procedure1A) was treated with trifluoroacetic acid (15 mL) at 25° C. for 1 hr.The solution was concentrated to dryness and treated with diethyl ether(10 mL). The solid was filtered and dried. It was dissolved in water(180 mL) and neutralized to pH=7.5 with saturated solution of sodiumbicarbonate. The mixture was stirred for 1 hr and the solid was filteredand dried giving the title compound (1.30 g) as white solid.

¹H NMR (DMSO-d₆) 1.76-1.85 (m, 1H), 1.99-2.08 (m, 1H), 2.65-2.81 (m,2H), 3.44 (dd, J=8 Hz, J=5 Hz, 1H), 7.15-7.31 (m, 6H), 7.54-7.58 (m,1H), 7.62-7.66 (m, 1H), 7.91-7.96 (m, 2H), 8.75 (d, J=2 Hz, 1H), 8.99(d, J=2 Hz, 1H)

(B) N-Boc trans-4-(N-Boc aminomethyl)-L-Prolinyl D-HomophenylalanineQuinoline-3 amide

A solution of N-Boc-trans-4-(N-Boc aminomethyl)-L-Proline (0.15 g, 0.44mmol, U.S. Pat. No. 6,399,629) and D-Homophenylalanine Quinoline-3-amide(0.23 g, 0.66 mmol) in ethyl acetate (10 mL) was treated withdicyclohexylcarbodiimide (0.12 g, 0.60 mmol). The reaction was stirredat 25° C. for 3 hr (HPLC monitoring RP-18, 250×4 mm,water:acetonitrile:triethylamine:acetic acid=500:500:0.6:1 (mL), UVdetector, λ=254 nm), filtered and the filtrate washed with 1Mhydrochloric acid (25 mL), saturated solution of sodium bicarbonate (25mL) and water (25 mL). The organic layer was dried over anhydrous sodiumsulfate, filtered and concentrated to dryness to afford crude compound.It was purified by column chromatography on silica gel (hexane:ethylacetate=1:3) to afford the title compound (0.22 g) as white solid.

¹H NMR (DMSO-d₆) (two rotamers) 1.36 (s, 18H), 1.38 (s, 18H), 1.88-1.92(m, 2H), 2.00-2.20 (m, 2H), 2.32-2.45 (m, 4H), 2.55-2.70 (m, 4H),2.89-2.98 (m, 5H), 3.05-3.09 (m, 1H) 3.30-3.34 (m, 2H), 3.46-3.51 (m,2H), 4.22-4.28 (m, 2H), 4.40-4.45 (m, 1H) 4.52-4.56 (m, 1H), 6.98-7.03(m, 2H), 7.16-7.30 (m, 10H), 7.55-7.66 (m, 4H), 7.90 (d, J=9 Hz, 2H),7.93-7.96 (dd, J=8 Hz, J=4 Hz, 2H), 8.36 (d, J=8 Hz, 1H), 8.54 (d, J=8Hz, 1H), 8.68 (dd, J=15 Hz, J=2 Hz, 1H), 8.94 (d, J=2 Hz, 1H), 8.99 (d,J=2 Hz, 1H), 10.06 (s, 1H), 10.62 (s, 1H)

(C) Amino-trans-4-aminomethyl-L-Prolinyl D-Homophenylalanine Quinoline-3amide Mesylate

N-Boc trans-4-(N-Boc aminomethyl)-L-Prolinyl D-HomophenylalanineQuinoline-3 amide was converted into mono-mesylate salt of the tileproduct according to the procedure described for compound 3 to give thetitle compound (0.11 g) as off white solid.

Example 4 Method I: 2-(S),5-Diamino-pentanoic acid[1-(R)-(quinolin-3-ylcarbamoyl)-2-(3,4,5-trifluoro-phenyl)-ethyl]-amide(Compound 41) (A) N-(Diphenylmethylene)(3,4,5-trifluoro)-D,L-Phenylalanine ethyl ester

A solution of N-(Diphenylmethylene)glycine ethyl ester (1.10 g, 4.1mmol) and 3,4,5-trifluorobenzylbromide (0.91 g, 4.0 mmol) indimethylformamide (10 mL) was treated with anhydrous potassium carbonate(2.83 g, 20.5 mmol) and stirred for 18 hrs at 25° C. The mixture wasfiltered and the filtrate was evaporated to dryness to give crudeproduct. It was purified by column chromatography on silica gel(hexane:ethyl acetate=4:1) to give title compound (1.2 g).

¹H NMR (DMSO-d₆) 1.17 (t, J=7 Hz, 3H), 3.05-3.19 (m, 2H), 4.06-4.18 (m,3H), 6.96-7.03 (m, 2H), 7.36-7.47 (m, 10H)

MS (EI) found for C₂₄H₂₀NO₂F₃ M⁺=411

(B) (3,4,5-trifluoro)-D,L-Phenylalanine hydrochloride

N-(Diphenylmethylene) (3,4,5-trifluoro)-D,L-Phenylalanine ethyl ester(1.33 g, 3.2 mmol) was suspended in water (15 mL) at 25° C. and treatedwith conc. hydrochloric acid (5 mL). The mixture was heated at refluxfor 4 hrs and evaporated to dryness. The residue was triturated withdiethyl ether (4×20 mL). The solid was filtered and dried to affordtitle compound (0.72 g)

¹H NMR (DMSO-d₆) 3.09-3.25 (m, 2H), 4.24 (t, J=7 Hz, 1H), 7.28-7.34 (m,2H), 8.50 (brs, 3H)

(C)N-Boc-(3,4,5-trifluoro)-D,L-Phenylalanine

(3,4,5-trifluoro)-D,L-Phenylalanine hydrochloride (0.72 g, 2.8 mmol) wassuspended in methylene chloride (20 mL), cooled to 0° C. and treatedwith triethylamine (2.0 mL, 14 mmol) and di-tert-butyl dicarbonate (0.68g, 3.1 mmol). The mixture was allowed to warm up to 25° C., stirred for18 hrs and evaporated to dryness. The residue was dissolved in water (50mL) and extracted with diethyl ether (2×20 mL). Water layer was cooledto 0° C. and acidified with 1M hydrochloric acid to pH=2. The mixturewas extracted with ethyl acetate (2×20 mL). Organic extract was driedover sodium sulfate, filtered and concentrated to give title compound(0.82 g).

¹H NMR (DMSO-d₆) 1.31 (s, 9H), 2.78 (dd, J=14 Hz, J=11 Hz, 1H), 3.04(dd, J=14 Hz, J=4 Hz, 1H) 4.09-4.15 (m, 1H), 7.13-7.23 (m, 3H)

(D) (3,4,5-trifluoro)-D,L-phenylalanine Quinoline-3-amideTrifluoroacetate

A solution of N-Boc-(3,4,5-trifluoro)-D,L-phenylalanine (0.81 g, 2.5mmol) and in dimethylformamide (10 mL) was treated with 3-aminoquinoline(0.55 g, 3.8 mmol) followed by dicyclohexylcarbodiimide (0.52 g, 2.5mmol). The reaction was stirred at 25° C. for 18 hr (HPLC monitoringRP-18, 250×4 mm, water:acetonitrile:triethylamineaceticacid=500:500:0.6:1 (mL), UV detector, λ=254 nm), filtered and thefiltrate evaporated to dryness. The residue was dissolved in methylenechloride (30 mL) and washed with 1M hydrochloric acid (25 mL), saturatedsolution of sodium bicarbonate (25 mL) and water (25 mL). The organiclayer was dried over anhydrous sodium sulfate, filtered and concentratedto dryness to afford Boc protected title compound (1.1 g) as whitesolid. It was treated with trifluoroacetic acid (10 mL) at 25° C. for 1hr. The solution was concentrated to dryness and treated with diethylether (10 mL). The solid was filtered and dried to give the titlecompound (1.31 g)

¹H NMR (DMSO-d₆) 3.13 (dd, J=14 Hz, J=8 Hz, 1H), 3.27 (dd, J=14 Hz, J=6Hz, 1H) 4.31 (brs, 1H), 7.23-7.31 (m, 2H), 7.61-7.64 (m, 1H), 7.69-7.73(m, 1H), 8.00 (d, J=8 Hz, 2H), 8.64 (d, J=2 Hz, 1H), 8.91 (d, J=2 Hz,1H), 11.04 (s, 1H)

MS (EI) found for C₂₃H₂₂N₃O₃F₃ M⁺=445

(E) N_(a)N_(d)-Bis-Boc-L-Ornithyl-(3,4,5-trifluoro)-D-PhenylalanineQuinoline-3-amide

A solution of N_(a)N_(d)-bis-Boc-L-Ornithine (1.25 g, 3.7 mmol),triethylamine (0.57 mL, 4.1 mmol) and methylene chloride (60 mL) wasstirred at 25° C. for 10 min, cooled to −10° C. and treated with ethylchloroformate (0.36 mL, 3.7 mmol). The mixture was stirred at −10° C.for 2 hrs. (3,4,5-trifluoro)-D,L-phenylalanine Quinoline-3-amideTrifluoroacetate (1.31 g) was suspended in methylene chloride (20 mL)and neutralized with triethylamine (0.70 mL, 5.0 mmol). The resultantsolution was added dropwise to the mixed anhydride and the mixture wasallowed to warm up to 25° C. and stirred for an additional 1 hr.Saturated sodium bicarbonate (30 mL) was added, the organic layer waswashed with water (30 mL), dried over anhydrous sodium sulfate, filteredand concentrated to dryness to afford crude product. TLC (silica gelMerck plates, diethyl ether:ethyl acetate=1:1) revealed the presence oftwo diastereoisomers. They were separated by column chromatography onsilica gel with diethyl ether:ethyl acetate=3:1 to give the titlecompound (0.25 g) as white solid.

¹H NMR (DMSO-d₆) 1.10-1.23 (m, 4H), 1.34 (s, 9H), 1.37 (s, 9H),2.78-2.83 (m, 2H), 2.88-2.94 (m, 1H), 3.20-3.25 (, 1H), 3.87-4.90 (m,1H) 4.75-4.78 (m, 1H), 6.70 (brs, 1H), 6.95 (d, J=7 Hz, 1H), 7.23 (dd,J=9 Hz, J=7 Hz, 2H), 7.57-7.61 (m, 1H), 7.64-7.69 (m, 1H), 7.92-7.98 (m,2H), 8.50 (d, J=8 Hz, 1H), 8.70 (d, J=2 Hz, 1H), 8.97 (d, J=2 Hz, 1H),10.36 (s, 1H)

MS found for C₃₃H₄₀N₅O₆F₃ (M+H)⁺=660

(F) L-ornithyl-(3,4,5-trifluoro)-D-phenylalanine Quinoline-3-amideMesylate

N_(a)N_(d)-bis-Boc-L-ornithyl-(3,4,5-trifluoro)-D-PhenylalanineQuinoline-3-amide was converted into mono-mesylate salt of the tileproduct according to the procedure described for compound 3 to give thetitle product (0.15 g) as off white solid.

Example 5 Method K:3-(S)-Amino-N-{4-(S)-amino-4-[3-phenyl-1-(R)-(quinolin-3-ylcarbamoyl)-propylcarbamoyl]-butyl}-succinamicacid mono-mesylate salt (compound 34) (A) N_(a)-Boc,N_(d)-Cbz-L-Ornithyl-D-Homophenylalanine Quinoline-3-amide

The title compound was prepared as in step (B) of Method Ha (compound 3)except that N_(a)-Boc, N_(d)-Cbz-L-Ornithine was used.

¹H NMR (DMSO-d₆) 1.35 (s, 9H), 1.40-1.71 (m, 4H) 1.90-1.99 (m, 1H),2.10-2.18 (m, 1H), 2.53-2.76 (m, 2H), 3.01 (q, J=6 Hz, 2H), 3.98-4.04(m, 1H), 4.44-4.49 (m, 1H), 4.98 (s, 2H), 7.08 (d, J=7 Hz, 1H),7.15-7.35 (m, 12H), 7.55-7.59 (m, 1H), 7.63-7.66 (m, 1H), 7.90-7.97 (m,2H), 8.45 (d, J=8 Hz, 1H), 8.70 (d, J=2 Hz, 1H), 8.98 (d, J=2 Hz, 1H),10.27 (s, 1H)

MS (EI) found for C₃₇H₄₃N₅O₆ M⁺=653

(B) N_(a)-Boc-L-Ornithyl-D-Homophenylalanine Quinoline-3-amide

A solution of N_(a)-Boc, N_(d)-Cbz-L-Ornithyl-D-HomophenylalanineQuinoline-3-amide (2.0 g, 3.06 mmol) in methanol (220 mL) was treatedwith 10% Pd/C catalyst (0.1 g) and stirred in the atmosphere of hydrogenat 40° C. for 3 hr. Catalyst was filtered through Celite pad and thefiltrate evaporated to dryness to give the title product (1.64 g)

¹H NMR (DMSO-d₆) 1.36 (s, 9H), 1.38-1.48 (m, 2H) 1.56-1.72 (m, 2H),1.92-2.02 (m, 1H), 2.10-2.18 (m, 1H), 2.55-2.76 (m, 4H), 3.98-4.02 (m,1H), 4.44-4.49 (m, 1H), 4.98 (s, 2H), 7.13 (d, J=7 Hz, 1H), 7.15-7.30(m, 5H), 7.55-7.59 (m, 1H), 7.63-7.66 (m, 1H), 7.90-7.96 (m, 2H), 8.52(d, J=8 Hz, 1H), 8.70 (d, J=2 Hz, 1H), 8.98 (d, J=2 Hz, 1H), 10.31 (s,1H)

MS found for C₂₉H₃₇N₅O₄ (M+H)⁺=520

(C) N-Boc-β-benzyl-L-Aspartyl N_(a)-Boc-L-Ornithyl-D-HomophenylalanineQuinoline-3-amide

A solution of N_(a)-Boc-L-Ornithyl-D-Homophenylalanine Quinoline-3-amide(0.8 g, 1.54 mmol) in methylene chloride (10 mL) was treated withN-Boc-β-benzyl-L-Aspartic acid N-hydroxysuccinimide ester (0.67 g, 1.60mmol) and stirred at 25° C. for 18 hr. The mixture was filtered and thefiltrate was concentrated to dryness. The residue was chromatographed onsilica gel (hexane:ethyl acetate=1:2) to give the title product (1.15g).

¹H NMR (DMSO-d₆) 1.36 (s, 18H), 1.40-1.64 (m, 4H) 1.85-1.89 (m, 1H),2.08-2.18 (m, 1H), 2.55-2.76 (m, 4H), 3.02-3.13 (m, 2H), 3.98-4.02 (m,1H), 4.29-4.33 (m, 1H), 4.45-4.49 (m, 1H), 5.06 (s, 2H), 7.06-7.09 (m,1H), 7.15-7.35 (m, 1H), 7.55-7.59 (m, 1H), 7.63-7.66 (m, 1H), 7.87-7.96(m, 3H), 8.40 (d, J=8 Hz, 1H), 8.70 (d, J=2 Hz, 1H), 8.98 (d, J=2 Hz,1H), 10.31 (s, 1H)

(D) N_(d)-L-aspartyl-L-Ornithyl-D-Homophenylalanine Quinoline-3-amide

A solution of N-Boc-β-benzyl-L-aspartylN_(a)-Boc-L-Ornithyl-D-Homophenylalanine Quinoline-3-amide (1.1 g, 1.33mmol) in methanol (200 mL) was treated with 10% Pd/C catalyst (0.1 g)and stirred in the atmosphere of hydrogen at 40° C. for 3 hr. Catalystwas filtered through Celite pad and the filtrate evaporated to dryness.The residue was converted into mono-mesylate salt of the tile productaccording to the procedure described for compound 3 to give the titlecompound.

Example 6 2-(R),5-Diamino-pentanoic acid[3-phenyl-1-(R)-(quinolin-3-ylcarbamoyl)-propyl]-amide (Compound 1)

2-(R),5-Diamino-pentanoic acid[3-phenyl-1-(R)-(quinolin-3-ylcarbamoyl)-propyl]-amide was obtainedaccording to Method Ha.

1H NMR 1.47-1.55 (m, 1H), 1.61-1.78 (m, 3H), 1.94-2.04 (m, 1H),2.08-2.17 (m, 1H), 2.60-2.68 (m, 1H), 2.71-2.84 (m, 3H), 3.39 (brs, 1H),4.56 (brs, 1H), 6.07 (brs, 4H), 7.16-7.31 (m, 5H), 7.55-7.60 (m, 1H),7.63-7.67 (m, 1H), 7.94 (dd, J=15 Hz, J=8 Hz, 2H), 8.46 (brs, 1H), 8.68(d, J=2 Hz, 1H) 8.94 (d, J=2 Hz, 1H), 10.62 (s, 1H)

MS C₂₄H₂₉N₅O₂ (M+H)⁺=420

Example 7 2-(S),5-Diamino-pentanoic acid[3-phenyl-1-(S)-(quinolin-3-ylcarbamoyl)-propyl]-amide (Compound 2)

2-(S),5-Diamino-pentanoic acid[3-phenyl-1-(S)-(quinolin-3-ylcarbamoyl)-propyl]-amide was obtainedaccording to Method Ha.

1H NMR 1.57-1.68 (m, 3H), 1.73-1.79 (m, 1H), 1.94-2.04 (m, 1H),2.08-2.17 (m, 1H), 2.59-2.67 (m, 1H), 2.70-2.76 (m, 1H), 2.80-2.85 (m,2H), 3.56 (brs, 1H), 4.57 (brs, 1H), 6.71 (brs, 4H), 7.16-7.31 (m, 5H),7.56-7.60 (m, 1H), 7.64-7.68 (m, 1H), 7.94 (dd, J=14 Hz, J=8 Hz, 2H),8.65 (brs, 1H), 8.67 (d, J=2 Hz 1H), 8.94 (d, J=2 Hz, 1H), 10.64 (s, 1H)

MS C₂₄H₂₉N₅O₂ (M+H)⁺=420

Example 8 2-(S),5-Diamino-pentanoic acid[3-phenyl-1-(R)-(quinolin-3-ylcarbamoyl)-propyl]-amide (Compound 3)

2-(S),5-Diamino-pentanoic acid[3-phenyl-1-(R)-(quinolin-3-ylcarbamoyl)-propyl]-amide was obtainedaccording to Method Ha.

1H NMR 1.48-1.57 (m, 1H), 1.62-1.77 (m, 2H), 1.94-2.04 (m, 1H),2.07-2.16 (m, 1H), 2.60-2.67 (m, 1H), 2.70-2.77 (m, 1H), 2.78-2.84 (m,2H), 3.39 (brs, 1H), 4.55 (brs, 1H), 6.03 (brs, 4H), 7.16-7.30 (m, 5H),7.55-7.60 (m, 1H), 7.63-7.67 (m, 1H), 7.94 (dd, J=14 Hz, J=8 Hz, 2H),8.50 (brs, 1H), 8.68 (d, J=2 Hz 1H), 8.95 (d, J=2 Hz, 1H), 10.61 (s, 1H)

MS C₂₄H₂₉N₅O₂ (M+H)⁺=420

Example 9 2-(R),5-Diamino-pentanoic acid[3-phenyl-1-(S)-(quinolin-3-ylcarbamoyl)-propyl]-amide (Compound 4)

2-(R),5-Diamino-pentanoic acid[3-phenyl-1-(S)-(quinolin-3-ylcarbamoyl)-propyl]-amide was obtainedaccording to Method Ha.

1H NMR 1.46-1.53 (m, 1H), 1.62-1.76 (m, 3H), 1.94-2.03 (m, 1H),2.07-2.16 (m, 1H), 2.60-2.68 (m, 1H), 2.70-2.84 (m, 3H), 3.37 (brs, 1H),4.56 (brs, 1H), 5.93 (brs, 4H), 7.16-7.31 (m, 5H), 7.55-7.61 (m, 1H),7.63-7.67 (m, 1H), 7.94 (dd, J=15 Hz, J=8 Hz, 2H), 8.43 (brs, 1H), 8.68(d, J=2 Hz, 1H), 8.94 (d, J=2 Hz, 1H), 10.61 (s, 1H)

MS C₂₄H₂₉N₅O₂ (M+H)⁺=420

Example 102-(S)-Amino-N-{4-amino-1-(S)-[3-phenyl-1-(R)-(quinolin-3-ylcarbamoyl)-propylcarbamoyl]-butyl}-succinamicacid (Compound 5)

2-(S)-Amino-N-{4-amino-1-(S)-[3-phenyl-1-(R)-(quinolin-3-ylcarbamoyl)-propylcarbamoyl]-butyl}-succinamicacid was obtained according to Method K.

1H NMR 1.43-1.53 (m, 1H), 1.55-1.70 (m, 2H), 1.80-1.90 (m, 1H),1.92-2.04 (m, 1H), 2.06-2.12 (m, 1H), 2.50-2.83 (m, 4H), 2.76-2.83 (m,2H), 4.06 (brs, 1H), 4.42 (brs, 1H), 4.52 (brs, 1H), 7.16-7.32 (m, 5H),7.55-7.60 (m, 1H), 7.62-7.66 (m, 1H), 7.88-7.95 (m, 2H), 8.65 (brs, 1H),8.70 (d, J=2 Hz 1H), 8.99 (d, J=2 Hz, 1H), 9.02 (d, J=2 Hz, 1H), 10.74(s, 1H)

MS C₂₈H₃₄N₆O₅ (M+H)⁺=535

Example 11 2-(S),5-Diamino-pentanoic acid[3-phenyl-1-(R)-(5,6,7,8-tetrahydro-quinolin-3-ylcarbamoyl)-propyl]-amide(Compound 6)

2-(S),5-Diamino-pentanoic acid[3-phenyl-1-(R)-(5,6,7,8-tetrahydro-quinolin-3-ylcarbamoyl)-propyl]-amidewas obtained according to Method Ha.

1H NMR 1.50-1.57 (m, 1H), 1.61-1.83 (m, 7H), 1.88-1.98 (m, 1H),2.00-2.09 (m, 1H), 2.55-2.61 (m, 1H), 2.64-2.75 (m, 5H), 2.78-2.84 (m,2H), 3.44 (brs, 1H), 4.47 (brs, 1H), 6.00 (brs, 4H), 7.17-7.30 (m, 5H),7.72 (d, J=2 Hz, 1H), 8.46 (d, J=2 Hz, 1H), 8.50 (brs, 1H), 10.18 (s,1H)

MS C₂₄H₃₃N₅O₂ (M+H)⁺=424

Example 12 2-(S),5-Diamino-pentanoic acid[3-phenyl-1-(R)-(quinolin-6-ylcarbamoyl)-propyl]-amide (Compound 7)

2-(S),5-Diamino-pentanoic acid[3-phenyl-1-(R)-(quinolin-6-ylcarbamoyl)-propyl]-amide was obtainedaccording to Method Ha.

1H NMR 1.51-1.60 (m, 1H), 1.60-1.69 (m, 2H), 1.71-1.79 (m, 1H),1.93-2.03 (m, 1H), 2.06-2.15 (m, 1H), 2.58-2.64 (m, 1H), 2.68-2.74 (m,1H), 2.78-2.85 (m, 2H), 3.43-3.47 (m, 1H), 4.54-4.57 (m, 1H), 6.19 (brs,4H) 7.17-7.31 (m, 5H), 7.49 (dd, J=8 Hz, J=4 Hz, 1H), 7.85 (dd, J=9 Hz,J=2 Hz, 1H), 7.98 (d, J=9 Hz, 1H), 8.28 (d, J=8 Hz, 1H), 8.36 (d, J=2Hz, 1H), 8.55 (brs, 1H), 8.79 (dd, J=4 Hz, J=1.5 Hz, 1H), 10.48 (s, 1H)

MS C₂₄H₂₉N₅O₂ (EI) M⁺=419

Example 13 2-(S),5-Diamino-pentanoic acid[3-(4-fluoro-phenyl)-1-(R)-(quinolin-3-ylcarbamoyl)-propyl]-amide(Compound 8)

2-(S),5-Diamino-pentanoic acid[3-(4-fluoro-phenyl)-1-(R)-(quinolin-3-ylcarbamoyl)-propyl]-amide wasobtained according to Method I.

1H NMR 1.57-1.79 (m, 4H), 1.93-2.03 (m, 1H), 2.07-2.15 (m, 1H),2.58-2.64 (m, 1H), 2.67-2.74 (m, 1H), 2.78-2.85 (m, 2H), 3.51 (brs, 1H),4.53 (brs, 1H), 7.08-7.12 (m, 2H), 7.23-7.29 (m, 2H) 7.56-7.60 (m, 1H),7.63-7.67 (m, 1H), 7.91-7.96 (m, 2H), 8.60 (brs, 1H), 8.67 (d, J=2 Hz,1H), 8.94 (d, J=2 Hz, 1H), 10.62 (s, 1H)

MS C₂₄H₂₈N₅O₂F (M+H)⁺=438

Example 14 2-(S),5-Diamino-pentanoic acid[3-(4-fluoro-phenyl)-1-(S)-(quinolin-3-ylcarbamoyl)-propyl]-amide(Compound 9)

2-(S),5-Diamino-pentanoic acid[3-(4-fluoro-phenyl)-1-(S)-(quinolin-3-ylcarbamoyl)-propyl]-amide wasobtained according to Method I.

1H NMR 1.51-1.57 (m, 1H), 1.62-1.75 (m, 3H), 1.93-2.03 (m, 1H),2.06-2.16 (m, 1H), 2.33 (s, 3H) 2.59-2.67 (m, 1H), 2.69-2.74 (m, 1H),2.78-2.85 (m, 2H), 3.43 (brs, 1H), 4.53 (brs, 1H), 7.07-7.12 (m, 2H),7.24-7.29 (m, 2H), 7.55-7.59 (m, 1H), 7.63-7.67 (m, 1H), 7.89-7.96 (m,2H), 8.48 (brs, 1H), 8.66 (d, J=2 Hz, 1H), 8.93 (d, J=2 Hz, 1H), 10.60(s, 1H)

MS C₂₄H₂₈N₅O₂F (M+H)⁺=438

Example 15 2-(S),5-Diamino-pentanoic acid[2-(4-fluoro-phenyl)-1-(R)-(quinolin-3-ylcarbamoyl)-ethyl]-amide(Compound 11)

2-(S),5-Diamino-pentanoic acid[2-(4-fluoro-phenyl)-1-(R)-(quinolin-3-ylcarbamoyl)-ethyl]-amide wasobtained according to Method Ha.

1H NMR 1.32-1.37 (m, 1H), 1.44-1.55 (m, 3H), 2.67-2.75 (m, 2H), 2.94(dd, J=13 Hz, J=9 Hz, 1H), 3.15 (dd, J=13 Hz, J=5 Hz, 1H), 3.32 (brs,1H), 4.79 (brs, 1H), 7.13 (t, J=9 Hz, 2H), 7.33 (dd, J=9 Hz, J=5 Hz,2H), 7.57-7.61 (m, 1H), 7.65-7.69 (m, 1H), 7.95 (t, J=9 Hz, 2H), 8.50(brs, 1H), 8.66 (d, J=2 Hz, 1H), 8.91 (d, J=2 Hz, 1H), 10.67 (s, 1H)

MS C₂₃H₂₆N₅O₂F (M+H)⁺=424

Example 16 2-(S),5-Diamino-pentanoic acid[1-(R)-(6-fluoro-quinolin-3-ylcarbamoyl)-3-phenyl-propyl]-amide(Compound 11)

2-(S),5-Diamino-pentanoic acid[1-(R)-(6-fluoro-quinolin-3-ylcarbamoyl)-3-phenyl-propyl]-amide wasobtained according to Method Ha.

1H NMR 1.54-1.60 (m, 1H), 1.62-1.69 (m, 2H), 1.71-1.76 (m, 1H),1.96-2.04 (m, 1H), 2.08-2.17 (m, 1H), 2.59-2.76 (m, 2H), 2.79-2.85 (m,2H), 3.47 (brs, 1H), 4.55 (brs, 1H), 7.17-7.31 (m, 5H), 7.52-7.58 (m,1H), 7.78 (dd, J=9 Hz, J=2 Hz, 1H), 8.02 (dd, J=9 Hz, J=5 Hz, 1H), 8.58(brs, 1H), 8.70 (d, J=2 Hz, 1H), 8.93 (d, J=2 Hz, 1H), 10.68 (s, 1H)

MS C₂₄H₂₈N₅O₂F (M+H)⁺=438

Example 17 2-(S),5-Diamino-pentanoic acid[1-(R)-(6-trifluoromethyl-quinolin-3-ylcarbamoyl)-3-phenyl-propyl]-amide(Compound 13)

2-(S),5-Diamino-pentanoic acid[1-(R)-(6-trifluoromethyl-quinolin-3-ylcarbamoyl)-3-phenyl-propyl]-amidewas obtained according to Method Ha.

1H NMR 1.56-1.69 (m, 3H), 1.72-1.78 (m, 1H), 1.96-2.05 (m, 1H),2.09-2.18 (m, 1H), 2.59-2.67 (m, 1H), 2.70-2.76 (m, 1H), 2.78-2.85 (m,2H), 3.54 (brs, 1H), 4.58 (brs, 1H), 7.16-7.31 (m, 5H), 7.89 (dd, J=9Hz, J=2 Hz, 1H), 8.16 (d, J=9 Hz, 1H), 8.49 (s, 1H), 8.65 (brs 1H), 8.87(d, J=2 Hz, 1H), 9.11 (d, J=2 Hz, 1H), 10.80 (s, 1H)

MS C₂₅H₂₈N₅O₂F₃ (M+H)⁺=488

Example 18 4-(R)-Aminomethyl-pyrrolidine-2-(S)-carboxylic acid[3-phenyl-1-(R)-(quinolin-3-ylcarbamoyl)-propyl]-amide (Compound 14)

4-(R)-Aminomethyl-pyrrolidine-2-(S)-carboxylic acid[3-phenyl-1-(R)-(quinolin-3-ylcarbamoyl)-propyl]-amide was obtainedaccording to Method Hb.

1H NMR 1.48-1.52 (m, 1H), 1.99-2.03 (m, 1H), 2.10-2.18 (m, 1H),2.58-2.73 (m, 4H), 2.83-2.86 (m, 3H), 3.17 (brs, 1H), 3.88 (brs, 1H),4.54-4.59 (m, 1H), 7.17-7.30 (m, 5H), 7.56-7.68 (m, 2H), 7.94 (dd, J=14Hz, J=8 Hz, 2H), 8.60 (brs, 1H), 8.68 (s 1H), 8.94 (d, J=2 Hz, 1H),10.66 (s, 1H)

MS C₂₅H₂₉N₅O₂ (M+H)⁺=432

Example 19 4-(S)-Aminomethyl-pyrrolidine-2-(S)-carboxylic acid[3-phenyl-1-(R)-(quinolin-3-ylcarbamoyl)-propyl]-amide (Compound 15)

4-(S)-Aminomethyl-pyrrolidine-2-(S)-carboxylic acid[3-phenyl-1-(R)-(quinolin-3-ylcarbamoyl)-propyl]-amide was obtainedaccording to Method Hb.

1H NMR 1.85-1.93 (m, 1H), 1.94-2.04 (m, 2H), 2.09-2.18 (m, 1H),2.58-2.78 (m, 4H), 2.81-2.85 (m, 2H), 3.12 (brs, 1H), 3.90 (brs, 1H),4.54-4.59 (m, 1H), 7.16-7.30 (m, 5H), 7.56-7.68 (m, 2H), 7.94 (dd, J=14Hz, J=8 Hz, 2H), 8.57 (brs, 1H), 8.68 (d, J=2 Hz 1H), 8.93 (d, J=2 Hz,1H), 10.66 (s, 1H)

MS C₂₅H₂₉N₅O₂ (M+H)⁺=432

Example 202-(S),4-Diamino-N-[3-phenyl-1-(R)-(quinolin-3-ylcarbamoyl)-propyl]-butyramide(Compound 16)

2-(S),4-Diamino-N-[3-phenyl-1-(R)-(quinolin-3-ylcarbamoyl)-propyl]-butyramidewas obtained according to Method Ha.

1H NMR 1.71-1.80 (m, 1H), 1.92-2.05 (m, 2H), 2.08-2.16 (m, 1H),2.59-2.67 (m, 1H), 2.69-2.77 (m, 1H), 2.93 (t, J=7 Hz, 2H), 3.57 (brs,1H), 4.53-4.57 (m, 1H), 7.16-7.31 (m, 5H), 7.55-7.59 (m, 1H), 7.63-7.67(m, 1H), 7.90-7.96 (m, 2H), 8.60 (brs, 1H), 8.67 (d, J=2 Hz, 1H), 8.94(d, J=2 Hz, 1H), 10.63 (s, 1H)

MS C₂₃H₂₇N₅O₂ (M+H)⁺=406

Example 21 2-(S),6-Diamino-hexanoic acid[3-phenyl-1-(R)-(quinolin-3-ylcarbamoyl)-propyl]-amide (Compound 17)

2-(S),6-Diamino-hexanoic acid[3-phenyl-1-(R)-(quinolin-3-ylcarbamoyl)-propyl]-amide was obtainedaccording to Method Ha.

1H NMR 1.35-1.46 (m, 2H), 1.52-1.58 (m, 3H), 1.67-1.74 (m, 1H),1.96-2.05 (m, 1H), 2.09-2.16 (m, 1H), 2.59-2.67 (m, 1H), 2.69-2.74 (m,1H), 2.77 (t, J=7 Hz, 2H), 3.52 (t, J=7 Hz, 1H), 4.54 (brs, 1H),7.17-7.31 (m, 5H), 7.56-7.60 (m, 1H), 7.63-7.68 (m, 1H), 7.91-7.97 (m,2H), 8.61 (brs, 1H), 8.68 (d, J=2 Hz, 1H), 8.95 (d, J=2 Hz, 1H), 10.61(s, 1H)

MS C₂₅H₃₁N₅O₂ (EI) M⁺=433

Example 22 2-(S),5-Diamino-pentanoic acid[1-(R)-(methyl-quinolin-3-yl-carbamoyl)-3-phenyl-propyl]-amide (Compound18)

2-(S),5-Diamino-pentanoic acid[1-(R)-(methyl-quinolin-3-yl-carbamoyl)-3-phenyl-propyl]-amide wasobtained according to Method Ha.

1H NMR 1.45-1.48 (m, 1H), 1.58-1.65 (m, 3H), 1.78-1.88 (m, 2H), 2.27(brs 1H), 2.48 (brs, 1H) 2.76-2.82 (m, 2H), 3.26 (s, 3H) 3.35 (brs, 1H),4.16 (brs, 1H), 5.90 (brs, 4H), 6.78-6.82 (m, 5H), 7.69 (t J=7 Hz, 1H),7.84 (t, J=7 Hz, 1H), 7.94 (d, J=8 Hz, 1H), 8.05 (d, J=8 Hz, 1H), 8.34(s, 1H), (8.41 (brs, 1H), 8.86 (s, 1H)

MS C₂₅H₃₁N₅O₂ (M+H)⁺=434

Example 23 2-(S),5-Diamino-pentanoic acid[3-phenyl-1-(R)-(quinoxalin-2-ylcarbamoyl)-propyl]-amide tristrifluoroacetate salt (Compound 19)

2-(S),5-Diamino-pentanoic acid[3-phenyl-1-(R)-(quinoxalin-2-ylcarbamoyl)-propyl]-amide tristrifluoroacetate salt was obtained according to Method Ha.

1H NMR 1.62-1.69 (m, 2H), 1.76-1.89 (m, 2H), 1.95-2.05 (m, 1H),2.11-2.19 (m, 1H), 2.62-2.79 (m, 2H), 2.83-2.89 (m, 2H), 3.97 (brs, 1H),4.75 (brs, 1H), 7.16-7.30 (m, 5H), 7.73-7.77 (m, 1H), 7.81-7.85 (m, 1H),7.89 (brs 2H) 7.90-7.92 (m, 1H), 8.05-8.07 (m, 1H), 8.28 (brs, 2H), 9.07(d, J=7 Hz, 1H), 9.60 (s, 1H), 11.45 (s, 1H)

MS C₂₅H₃₁N₅O₂ (M+H)⁺=434

Example 24 2-(S),5-Diamino-pentanoic acid[1-(R)-(quinolin-3-ylcarbamoyl)-3-(4-trifluoromethyl-phenyl)-propyl]-amidetris trifluoroacetate salt (Compound 20)

2-(S),5-Diamino-pentanoic acid[1-(R)-(quinolin-3-ylcarbamoyl)-3-(4-trifluoromethyl-phenyl)-propyl]-amidetris trifluoroacetate salt was obtained according to Method Ha.

1H NMR 1.61-1.68 (m, 2H), 1.74-1.83 (m, 2H), 1.98-2.08 (m, 1H),2.15-2.23 (m, 1H), 2.73-2.91 (m, 4H), 3.93-4.01 (m, 1H), 4.57-4.63 (m,1H), 7.45-7.48 (m, 2H), 7.56-7.60 (m, 1H), 7.63-7.68 (m, 3H), 7.78 (brs3H), 7.89-7.96 (m, 2H), 8.27 (brs, m 3H), 8.64 (d, J=2 Hz, 1H),8.93-8.97 (m, 3H), 10.73 (s, 1H)

Example 25 2-(S),5-Diamino-pentanoic acid[1-(R)-(quinolin-3-ylcarbamoyl)-2-(4-trifluoromethyl-phenyl)-ethyl]-amide(Compound 21)

2-(S),5-Diamino-pentanoic acid[1-(R)-(quinolin-3-ylcarbamoyl)-2-(4-trifluoromethyl-phenyl)-ethyl]-amidewas obtained according to Method Ha.

1H NMR 1.28-1.35 (m, 1H), 1.44-1.52 (m, 3H), 2.65-2.74 (m, 2H), 3.06(dd, J=13 Hz, J=9 Hz, 1H), 3.27 (dd, J=13 Hz, J=5 Hz, 1H), 3.34 (brs,1H), 4.86 (brs, 1H), 7.52-7.69 (m, 6H), 8.54 (brs, 1H), 8.66 (d, J=2 Hz,1H), 8.92 (d, J=2 Hz, 1H), 10.72 (s, 1H)

MS C₂₄H₂₆N₅O₂F₃ (M+H)⁺=474

Example 26 2(R)-(2-(S),5-Diamino-pentanoylamino)-5-methyl-hexanoic acidquinolin-3-ylamide (Compound 22)

2(R)-(2-(S),5-Diamino-pentanoylamino)-5-methyl-hexanoic acidquinolin-3-ylamide was obtained according to Method I.

1H NMR 0.87 (dd, J=7 Hz, J=2 Hz, 6H), 1.16-1.32 (m, 2H), 1.51-1.74 (m,6H), 1.77-1.86 (m, 1H), 2.75-2.86 (m, 2H), 3.50 (brs, 1H), 4.48-4.55 (m,1H), 6.51 (brs, 4H), 7.56-7.68 (m, 2H), 7.92-7.97 (m, 2H), 8.50 (brs1H), 8.69 (d, J=2 Hz, 1H), 8.96 (d, J=2 Hz, 1H), 10.63 (s, 1H)

MS C₂₁H₃₁N₅O₂ (M+H)⁺=386

Example 27 2-(S),5-Diamino-pentanoic acid[2-phenyl-1-(R)-(quinolin-3-ylcarbamoyl)-ethyl]-amide (Compound 23)

2-(S),5-Diamino-pentanoic acid[2-phenyl-1-(R)-(quinolin-3-ylcarbamoyl)-ethyl]-amide was obtainedaccording to Method Ha.

1H NMR 1.31-1.38 (m, 1H), 1.42-1.55 (m, 3H), 2.63-2.75 (m, 2H), 2.95(dd, J=13 Hz, J=9 Hz, 1H), 3.17 (dd, J=13 Hz, J=5 Hz, 1H), 3.37 (brs,1H), 4.81 (brs, 1H), 7.18-7.31 (m, 5H), 7.56-7.60 (m, 1H), 7.63-7.69 (m,1H), 7.91-7.97 (m, 2H), 8.53 (brs, 1H), 8.65 (d, J=2 Hz, 1H), 8.90 (d,J=2 Hz, 1H), 10.65 (s, 1H)

MS C₂₃H₂₇N₅O₂ (M+H)⁺=406

Example 28 2-(S),5-Diamino-pentanoic acid[1-(R)-(3,4-dihydro-1H-isoquinoline-2-carbonyl)-3-phenyl-propyl]-amide(Compound 24)

2-(S),5-Diamino-pentanoic acid[1-(R)-(3,4-dihydro-1H-isoquinoline-2-carbonyl)-3-phenyl-propyl]-amidewas obtained according to Method Ha.

1H NMR 1.41-1.48 (m, 1H), 1.62-1.68 (m, 3H), 1.80-1.96 (m, 2H),2.53-2.64 (m, 2H), 2.74-2.81 (m, 2H), 3.25-3.28 (m, 1H), 3.52-3.74 (m,3H), 4.50-4.56 (m, 3H), 4.80-4.85 (m, 1H), 7.13-7.30 (m, 9H), 8.30-8.35(m, 2H)

MS C₂₄H₃₂N₄O₂ (M+H)⁺=409

Example 29 2-(S),5-Diamino-pentanoic acid[1-(R)-(biphenyl-4-ylcarbamoyl)-3-phenyl-propyl]-amide (Compound 25)

2-(S),5-Diamino-pentanoic acid[1-(R)-(biphenyl-4-ylcarbamoyl)-3-phenyl-propyl]-amide was obtainedaccording to Method Ha.

1H NMR 1.60-1.68 (m, 2H), 1.72-1.87 (m, 2H), 1.94-2.00 (m, 1H),2.05-2.12 (m, 1H), 2.55-2.73 (m, 2H), 2.82-2.88 (m, 2H), 3.85-3.89 (m,1H), 4.56-4.62 (m, 1H), 7.20-7.74 (m, 14H), 9.00 (d, J=8 Hz, 1H), 10.36(s, 1H)

MS C₂₇H₃₂N₄O₂ (M+H)⁺=445

Example 30 2-(S),5-Diamino-pentanoic acid[1-(R)-(biphenyl-3-ylcarbamoyl)-3-phenyl-propyl-amide (Compound 26)

2-(S),5-Diamino-pentanoic acid[1-(R)-(biphenyl-3-ylcarbamoyl)-3-phenyl-propyl-amide was obtainedaccording to Method Ha.

1H NMR 1.60-1.66 (m, 2H), 1.73-1.82 (m, 2H), 1.92-2.00 (m, 1H),2.05-2.14 (m, 1H), 2.56-2.72 (m, 2H), 2.84 (t, J=7 Hz, 2H), 3.86 (brs,1H), 4.54-4.60 (m, 1H), 7.18-7.72 (m, 13H), 7.93 (s, 1H), 8.90 (d, J=8Hz, 1H), 10.33 (s, 1H)

MS C₂₇H₃₂N₄O₂ (M+H)⁺=445

Example 31 2-(S),5-Diamino-pentanoic acid[3-phenyl-1-(R)-(quinolin-7-ylcarbamoyl)-propyl]-amide (Compound 28)

2-(S),5-Diamino-pentanoic acid[3-phenyl-1-(R)-(quinolin-7-ylcarbamoyl)-propyl]-amide was obtainedaccording to Method Ha.

1H NMR 1.51-1.59 (m, 1H), 1.62-1.69 (m, 2H), 1.70-1.77 (m, 1H),1.93-2.03 (m, 1H), 2.06-2.13 (m, 1H), 2.57-2.65 (m, 1H), 2.67-2.76 (m,1H), 2.77-2.85 (m, 2H), 3.46 (brs, 1H), 4.58 (brs, 1H), 7.19-7.30 (m,5H), 7.42 (dd, J=8 Hz, J=4 Hz, 1H), 7.73 (dd, J=9 Hz, J=2 Hz, 1H), 7.93(d, J=9 Hz, 1H), 8.27 (d, J=8 Hz, 1H), 8.45 (d, J=2 Hz, 1H), 8.55 (brs,1H), 8.84 (dd, J=4 Hz, J=1.5 Hz, 1H), 10.51 (s, 1H)

MS C₂₄H₂₉N₅O₂ (M+H)⁺=420

Example 32 2-(S),5-Diamino-pentanoic acid[1-(R)-(2-fluoro-5-trifluoromethyl-phenylcarbamoyl)-3-phenyl-propyl]-amide(Compound 29)

2-(S),5-Diamino-pentanoic acid[1-(R)-(2-fluoro-5-trifluoromethyl-phenylcarbamoyl)-3-phenyl-propyl]-amidewas obtained according to Method Ha.

1H NMR 1.62-1.72 (m, 2H), 1.76-1.90 (m, 2H), 1.94-2.02 (m, 1H),2.04-2.12 (m, 1H), 2.59-2.74 (m, 2H), 2.80-2.87 (m, 2H), 3.95 (brs, 1H),4.68 (brs, 1H), 7.17-7.33 (m, 6H), 7.51-7.60 (m, 2H), 7.90 (brs, 2H,NH), 8.28 (brs, 2H, NH) 9.10 (d, J=7 Hz, 1H), 10.34 (s, 1H)

MS C₂₂H₂₆N₄O₂F₄ (M+H)⁺=455

Example 33 2-(S),5-Diamino-pentanoic acid[3-phenyl-1-(R)-(3,4,5-trifluoro-phenylcarbamoyl)-propyl]-amide(Compound 30)

2-(S),5-Diamino-pentanoic acid[3-phenyl-1-(R)-(3,4,5-trifluoro-phenylcarbamoyl)-propyl]-amide wasobtained according to Method Ha.

1H NMR 1.52-1.75 (m, 4H), 1.88-1.97 (m, 1H), 1.99-2.07 (m, 1H),2.53-2.61 (m, 1H), 2.64-2.71 (m, 1H), 2.76-2.84 (m, 2H), 3.47 (brs, 1H),4.41 (brs, 1H), 7.16-7.29 (m, 5H), 7.53 (dd, J=10 Hz, J=6 Hz, 2H), 8.56(brs, 1H), 10.52 (s, 1H)

MS C₂₁H₂₅N₄O₂F₃ (M+H)⁺=423

Example 34 2-(S),5-Diamino-pentanoic acid[3-phenyl-1-(R)-(2,3,4-trifluoro-phenylcarbamoyl)-propyl]-amide(Compound 31)

2-(S),5-Diamino-pentanoic acid[3-phenyl-1-(R)-(2,3,4-trifluoro-phenylcarbamoyl)-propyl]-amide wasobtained according to Method Ha.

1H NMR 1.55-1.75 (m, 4H), 1.90-1.98 (m, 1H), 2.01-2.09 (m, 1H),2.56-2.72 (m, 2H), 2.76-2.82 (m, 2H), 3.48 (brs, 1H), 4.59 (brs, 1H),7.17-7.33 (m, 6H), 7.47-7.53 (m, 1H), 8.56 (brs, 1H), 10.52 (s, 1H)

MS C₂₁H₂₅N₄O₂F₃ (M+H)⁺=423

Example 35 2-(S),5-Diamino-pentanoic acid[1-(R)-(5-chloro-2-fluoro-phenylcarbamoyl)-3-phenyl-propyl]-amide(Compound 32)

2-(S),5-Diamino-pentanoic acid[1-(R)-(5-chloro-2-fluoro-phenylcarbamoyl)-3-phenyl-propyl]-amide wasobtained according to Method Ha.

1H NMR 1.58-1.75 (m, 4H), 1.87-1.97 (m, 1H), 2.01-2.10 (m, 1H),2.58-2.72 (m, 2H), 2.76-2.82 (m, 2H), 3.54 (brs, 1H), 4.65 (brs, 1H),7.17-7.36 (m, 7H), 7.97 (dd, J=7 Hz, J=2 Hz, 1H), 8.60 (brs, 1H), 10.12(s, 1H)

MS C₂₁H₂₆N₄O₂F³⁵Cl (M+H)⁺=421

Example 36 2-(S),5-Diamino-pentanoic acid[1-(R)-(1-methyl-2-oxo-1,2-dihydro-quinolin-3-ylcarbamoyl)-3-phenyl-propyl]-amide(Compound 33)

2-(S),5-Diamino-pentanoic acid[1-(R)-(1-methyl-2-oxo-1,2-dihydro-quinolin-3-ylcarbamoyl)-3-phenyl-propyl]-amidewas obtained according to Method Ha.

1H NMR 1.54-1.77 (m, 4H), 1.90-2.01 (m, 1H), 2.07-2.17 (m, 1H),2.58-2.76 (m, 2H), 2.78-2.85 (m, 2H), 3.57 (brs, 1H), 3.73 (s, 3H),4.52-4.56 (m, 1H), 5.95 (brs, 4H), 7.16-7.31 (m, 7H), 7.55-7.58 (m, 2H),7.71 (d, J=8 Hz, 1H), 8.62 (s, 1H), 9.55 (s, 1H)

MS C₂₅H₃₁N₅O₃ (M+H)⁺=450

Example 373-(S)-Amino-N-{4-(S)-amino-4-[3-phenyl-1-(R)-(quinolin-3-ylcarbamoyl)-propylcarbamoyl]-butyl}-succinamicacid acid (Compound 34)

3-(S)-Amino-N-{4-(S)-amino-4-[3-phenyl-1-(R)-(quinolin-3-ylcarbamoyl)-propylcarbamoyl]-butyl}-succinamicacid acid was obtained according to Method K.

1H NMR 1.48-1.56 (m, 1H), 1.60-1.70 (m, 2H), 1.82-1.90 (m, 1H),2.07-2.17 (m, 2H), 2.49-2.55 (m, 2H), 2.60-2.67 (m, 1H), 2.70-2.77 (m,1H), 2.99-3.05 (m, 1H), 3.33-3.40 (m, 1H), 3.77-3.82 (m, 2H), 4.43-4.48(m, 1H), 7.16-7.32 (m, 5H), 7.55-7.60 (m, 1H), 7.62-7.66 (m, 1H),7.90-7.96 (m, 2H), 8.50 (brs, 1H), 8.70 (d, J=2 Hz 1H), 9.00 (d, J=2 Hz,1H), 9.61 (d, J=7 Hz, 1H), 11.02 (s, 1H)

MS C₂₈H₃₄N₆O₅ (M+H)⁺=535

Example 382-(R)-[2-(S)-Amino-3-(2-amino-ethoxy)-propionylamino]-4-phenyl-N-quinolin-3-yl-butyramide(Compound 35)

2-(R)-[2-(S)-Amino-3-(2-amino-ethoxy)-propionylamino]-4-phenyl-N-quinolin-3-yl-butyramidewas obtained according to Method Hb.

1H NMR 1.96-2.05 (m, 1H), 2.08-2.17 (m, 1H), 2.58-2.79 (m, 2H), 2.95 (t,J=5 Hz, 2H), 3.50-3.65 (m, 4H), 3.74 (brs, 1H), 4.55 (brs, 1H),7.16-7.31 (m, 7H), 7.55-7.58 (m, 2H), 7.90-7.96 (m, 12H), 8.40 (brs 1H)8.67 (d, J=2 Hz, 1H), 8.93 (d, J=2 Hz, 1H), 10.61 (s, 1H)

MS C₂₄H₂₉N₅O₃ (M+H)⁺=436

Example 39 2-(S),5-Diamino-pentanoic acid[1-(R)-(3,5-dichloro-pyridin-2-ylcarbamoyl)-3-phenyl-propyl]-amide(Compound 36)

2-(S),5-Diamino-pentanoic acid[1-(R)-(3,5-dichloro-pyridin-2-ylcarbamoyl)-3-phenyl-propyl]-amide wasobtained according to Method Hb.

1H NMR 1.58-1.76 (m, 4H), 1.90-2.00 (m, 1H), 2.02-2.13 (m, 1H),2.60-2.83 (m, 4H), 3.63 (brs, 1H), 4.62 (brs, 1H), 7.18-7.30 (m, 5H),8.31 (d, J=2 Hz, 1H), 8.49 (d, J=2 Hz, 1H), 8.67 (brs, 1H), 10.58 (brs,1H)

MS C₂₀H₂₅N₅O₂ 35Cl2 (M+H)⁺=438

Example 40 2-(S),5-Diamino-pentanoic acid[1-(R)-(5-fluoro-2-hydroxy-phenylcarbamoyl)-3-phenyl-propyl]-amide(Compound 37

2-(S),5-Diamino-pentanoic acid[1-(R)-(5-fluoro-2-hydroxy-phenylcarbamoyl)-3-phenyl-propyl]-amide wasobtained according to Method Hb.

1H NMR 1.48-1.72 (m, 4H), 1.87-1.97 (m, 1H), 2.02-2.11 (m, 1H),2.55-2.72 (m, 2H), 2.76-2.82 (m, 2H), 3.50 (brs, 1H), 4.57 (brs, 1H),6.72-6.77 (m, 1H), 6.81-6.85 (m, 1H), 7.16-7.30 (m, 5H), 7.80 (dd, J=11Hz, J=3 Hz, 1H), 8.46 (brs, 1H), 9.31 (brs, 1H)

MS C₂₁H₂₇N₄O₃F (M+H)⁺=403

Example 41 2-(S),5-Diamino-pentanoic acid[1-(R)-(cinnolin-3-ylcarbamoyl)-3-phenyl-propyl]amide tristrifluoroacetate salt (Compound 39)

2-(S),5-Diamino-pentanoic acid[1-(R)-(cinnolin-3-ylcarbamoyl)-3-phenyl-propyl]-amide tristrifluoroacetate salt was obtained according to Method Ha.

1H NMR 1.60-1.69 (m, 2H), 1.77-1.87 (m, 2H), 1.95-2.05 (m, 1H),2.11-2.19 (m, 1H), 2.61-2.79 (m, 2H), 2.83-2.89 (m, 2H), 3.95 (brs, 1H),4.78-4.83 (m, 1H), 7.16-7.30 (m, 5H), 7.82-7.84 (m, 4H), 8.03-8.07 (m,1H), 8.25 (brs, 2H), 8.38-8.42 (m, 1H), 8.71 (s, 1H), 9.05 (d, J=8 Hz,1H), 11.71 (s, 1H)

MS C₂₃H₂₈N₆O₂ (M+H)⁺=421

Example 42 2-(S),5-Diamino-pentanoic acid[1-(R)-(2-oxo-1,2-dihydro-quinolin-3-ylcarbamoyl)-3-phenyl-propyl]-amide(Compound 40)

2-(S),5-Diamino-pentanoic acid[1-(R)-(2-oxo-1,2-dihydro-quinolin-3-ylcarbamoyl)-3-phenyl-propyl]-amidewas obtained according to Method Ha.

1H NMR 1.54-1.74 (m, 4H), 1.92-2.00 (m, 1H), 2.06-2.15 (m, 1H),2.58-2.74 (m, 2H), 2.79-2.83 (m, 2H), 3.39 (brs, 1H), 4.57 (brs, 1H),7.17-7.32 (m, 7H), 7.41-7.44 (m, 1H), 7.64 (d, J=8 Hz, 1H), 8.62 (s,1H), 9.52 (s, 1H), 12.32 (brs, 1H)

MS C₂₄H₂₉N₅O₃ (M+H)⁺=436

Example 43 2-(S),5-Diamino-pentanoic acid[1-(R)-(quinolin-3-ylcarbamoyl)-2-(3,4,5-trifluoro-phenyl)-ethyl]-amide(Compound 41)

2-(S),5-Diamino-pentanoic acid[1-(R)-(quinolin-3-ylcarbamoyl)-2-(3,4,5-trifluoro-phenyl)-ethyl]-amidewas obtained according to Method I.

1H NMR 1.27-1.36 (m, 1H), 1.46-1.53 (m, 3H), 2.66-2.76 (m, 2H), 2.96(dd, J=13 Hz, J=9 Hz, 1H), 3.16 (dd, J=13 Hz, J=4 Hz, 1H), 3.24 (brs,1H), 4.76-4.80 (m, 1H), 7.26 (dd, J=9 Hz, J=7 Hz, 2H), 7.57-7.61 (m,1H), 7.65-7.69 (m, 1H), 7.93-7.98 (m, 2H), 8.47 (brs, 1), 8.66 (d, J=2Hz, 1H), 8.93 (d, J=2 Hz, 1H), 10.65 (s, 1H)

MS C₂₃H₂₄N₅O₂F₃ (M+H)⁺=460

Example 44-Amino-(S)—N-{4-(S)-(2-(3)-(S)-amino-3-carboxy-propionylamino)-4-[3-phenyl-1-(R)-(quinolin-3-ylcarbamoyl)-propylcarbamoyl]-butyl}-succinamicacid tris methanesulfonate (Compound 43)

3-Amino-(S)—N-{4-(S)-(2-(3)-(S)-amino-3-carboxy-propionylamino)-4-[3-phenyl-1-(R)-(quinolin-3-ylcarbamoyl)-propylcarbamoyl]-butyl}-succinamicacid tris methanesulfonate was obtained according to Method L.

1H NMR 1.44-1.64 (m, 3H), 1.74-1.82 (m, 1H), 1.93-2.03 (m, 1H),2.07-2.16 (m, 1H), 2.53-2.94 (m, 6H), 3.09-3.21 (m, 1H), 3.46-3.58 (m,1H), 4.02 (brs, 1H), 4.17 (brs, 1H), 4.39-4.44 (m, 1H), 4.47-4.53 (m,1H), 7.16-7.32 (m, 5H), 7.55-7.60 (m, 1H), 7.62-7.66 (m, 1H), 7.90-7.96(m, 2H), 8.15 (brs, 4H), 8.44 (t, J=6 Hz, 1H), 8.51 (d, J=8 Hz, 1H),8.69 (d, J=2 Hz 1H), 8.97 (d, J=2 Hz, 1H), 10.53 (s, 1H), 12.95 (brs,2H)

MS C₃₂H₃₉N₇O₈ (M+H)⁺=650

Example 45 5-Amino-2-(S)-methylamino-pentanoic acid[3-phenyl-1-(R)-(quinolin-3-ylcarbamoyl)-propyl]-amide (Compound 45)

5-Amino-2-(S)-methylamino-pentanoic acid[3-phenyl-1-(R)-(quinolin-3-ylcarbamoyl)-propyl]-amide was obtainedaccording to Method Hb.

1H NMR 1.57-1.61 (m, 2H), 1.70-1.79 (m, 2H), 2.03-2.07 (m, 1H),2.10-2.15 (m, 1H), 2.55 (s, 3H), 2.65-2.75 (m, 2H), 2.84-2.88 (m, 2H),3.86 (brs, 1H), 4.56 (brs, 1H), 7.20-7.32 (m, 5H), 7.56-7.68 (m, 2H),7.65 (brs, 1H), 8.66 (d, J=2 Hz 1H), 8.95 (d, J=2 Hz, 1H), 10.73 (s, 1H)

MS C₂₅H₃₁N₅O₂ (M+H)⁺=434

Example 46 2-(S),5-Diamino-pentanoic acid[1-(R)-(6-fluoro-quinolin-3-ylcarbamoyl)-2-(4trifluoromethyl-phenyl)-ethyl]-amide (Compound 46)

2-(S),5-Diamino-pentanoic acid[1-(R)-(6-fluoro-quinolin-3-ylcarbamoyl)-2-(4-trifluoromethyl-phenyl)-ethyl]-amidewas obtained according to Method Ha.

1H NMR 1.27-1.34 (m, 1H), 1.42-1.50 (m, 3H), 2.62-2.75 (m, 2H), 3.05(dd, J=13 Hz, J=9 Hz, 1H), 3.26 (dd, J=13 Hz, J=5 Hz, 1H), 3.34 (brs,1H), 4.84-4.88 (m, 1H), 7.52-7.58 (m, 3H), 7.67 (d, J=8 Hz, 2H), 7.79(dd, J=10 Hz, J=3 Hz, 1H), 8.02 (dd, J=9 Hz, J=5 Hz, 1H), 8.54 (brs,1H), 8.68 (d, J=2 Hz, 1H), 8.90 (d, J=2 Hz, 1H), 10.79 (s, 1H)

MS C₂₄H₂₅N₅O₂F₄ (M+H)⁺=492

Example 47 2-(S),5-Diamino-pentanoic acid[1-(R)-(benzothiazol-6-ylcarbamoyl)-3-phenyl-propyl]-amide (Compound 50)

2-(S),5-Diamino-pentanoic acid[1-(R)-(benzothiazol-6-ylcarbamoyl)-3-phenyl-propyl]-amide was obtainedaccording to Method Ha.

1H NMR 1.54-1.75 (m, 4H), 1.90-2.00 (m, 1H), 2.03-2.12 (m, 1H),2.56-2.73 (m, 2H), 2.79-2.85 (m, 2H), 3.50 (brs, 1H), 4.55 (brs, 1H),6.41 (brs, 4H), 7.16-7.31 (m, 5H), 7.65 (dd, J=9 Hz, J=2 Hz, 1H), 8.02(d, J=9 Hz, 1H), 8.52 (d, J=2 Hz, 1H), 8.55 (brs, 1H), 9.27 (s, 1H),10.43 (s, 1H)

MS C₂₂H₂₇N₅O₂S (M+H)⁺=426

Example 48 4-(R)-Aminomethyl-pyrrolidine-2-(S)-carboxylic acid[3-phenyl-1-(R)-(quinolin-6-ylcarbamoyl)-propyl]-amide tristrifluoroacetate salt (Compound 52)

4-(R)-Aminomethyl-pyrrolidine-2-(S)-carboxylic acid[3-phenyl-1-(R)-(quinolin-6-ylcarbamoyl)-propyl]-amide tristrifluoroacetate salt was obtained according to Method Hb.

1H NMR 1.60-1.68 (m, 1H), 1.94-2.04 (m, 1H), 2.09-2.18 (m, 1H),2.52-2.74 (m, 4H), 2.92-3.04 (m, 3H), 3.43 (brs, 1H), 4.30-4.37 (m, 1H),4.56-4.61 (m, 1H), 7.18-7.32 (m, 5H), 7.57 (dd, J=8 Hz, J=4 Hz, 1H),7.86-8.02 (m, 5H), 8.38-8.42 (m, 2H), 8.74 (brs, 1H), 8.86 (dd, J=4 Hz,J=2 Hz 1H), 9.13 (d, J=8 Hz, 1H), 9.52 (brs 1H), 10.65 (s, 1H)

MS C₂₅H₂₉N₅O₂ (M+H)⁺=432

Example 49 4-(S)-Aminomethyl-pyrrolidine-2-(S)-carboxylic acid[3-phenyl-1-(R)-(quinolin-6-ylcarbamoyl)-propyl]-amide tristrifluoroacetate salt (Compound 53)

4-(S)-Aminomethyl-pyrrolidine-2-(S)-carboxylic acid[3-phenyl-1-(R)-(quinolin-6-ylcarbamoyl)-propyl]-amide tristrifluoroacetate salt was obtained according to Method Hb.

1H NMR 1.94-2.03 (m, 1H), 2.09-2.22 (m, 3H), 2.52-2.76 (m, 3H), 2.96 (t,J=6 Hz, 2H), 3.05 (brs, 1H), 3.51 (brs, 1H), 4.46 (brs 1H), 4.54-4.60(m, 1H), 7.18-7.32 (m, 5H), 7.59 (dd, J=8 Hz, J=4 Hz, 1H), 7.89-8.05 (m,5H), 8.41-8.44 (m, 2H), 8.77 (brs, 1H), 8.87 (dd, J=4 Hz, J=2 Hz 1H),9.16 (d, J=8 Hz, 1H), 9.78 (brs 1H), 10.66 (s, 1H)

MS C₂₅H₂₉N₅O₂ (M+H)⁺=432

Example 50 2-(S)-Amino-6-methylamino-hexanoic acid[3-phenyl-1-(R)-(quinolin-3-ylcarbamoyl)-propyl]-amide (Compound 55)

2-(S)-Amino-6-methylamino-hexanoic acid[3-phenyl-1-(R)-(quinolin-3-ylcarbamoyl)-propyl]-amide was obtainedaccording to Method Hb.

1H NMR 1.34-1.43 (m, 2H), 1.50-1.58 (m, 3H), 1.65-1.73 (m, 1H),1.96-2.04 (m, 1H), 2.09-2.16 (m, 1H), 2.58-2.76 (m, 2H), 2.81 (t, J=7Hz, 2H), 3.25-3.40 (m, 3H), 3.43 (brs, 1H), 4.54 (brs, 1H), 7.17-7.31(m, 5H), 7.56-7.60 (m, 1H), 7.63-7.68 (m, 1H), 7.91-7.97 (m, 2H), 8.58(brs, 1H), 8.68 (d, J=2 Hz, 1H), 8.95 (d, J=2 Hz, 1H), 10.61 (s, 1H)

MS C₂₆H₃₃N₅O₃ (M+H)⁺=448

Example 51 2,5-Diamino-pentanoic acid[3-phenyl-1-(R)-(5-phenyl-[1,3,4]thiadiazol-2-ylcarbamoyl)-propyl]-amide(Compound 58)

2,5-Diamino-pentanoic acid[3-phenyl-1-(R)-(5-phenyl-[1,3,4]thiadiazol-2-ylcarbamoyl)-propyl]-amidewas obtained according to Method Ha.

1H NMR 1.58-1.77 (m, 4H), 1.93-2.02 (m, 1H), 2.04-2.14 (m, 1H),2.56-2.73 (m, 2H), 2.82 (t, J=7 Hz, 2H), 3.54 (brs, 1H), 4.56 (brs, 1H),7.1.6-7.30 (m, 6H), 7.51-7.54 (m, 3H), 7.90-7.93 (m, 2H), 8.63 (brs, 1H)

MS C₂₃H₂₈N₆O₂S (M+H)⁺=453

Example 52 2-(S),5-Diamino-pentanoic acid[3-phenyl-1-(R)-(4-phenyl-thiazol-2-ylcarbamoyl)-propyl]-amide (Compound59)

2-(S),5-Diamino-pentanoic acid[3-phenyl-1-(R)-(4-phenyl-thiazol-2-ylcarbamoyl)-propyl]-amide wasobtained according to Method F.

1H NMR 1.47-1.55 (m, 1H), 1.63-1.75 (m, 3H), 1.92-2.02 (m, 1H),2.08-2.17 (m, 1H), 2.55-2.62 (m, 1H), 2.76-2.84 (m, 2H), 3.40 (brs, 1H),4.59 (brs, 1H), 6.27 (brs, 4H), 7.16-7.34 (m, 8H), 7.41-7.44 (m, 2H),7.63 (m, 1H), 7.88 (d, J=1 Hz, 1H), 7.81 (d, J=2 Hz, 1H)

MS C₂₄H₂₉N₅O₂S (M+H)⁺=452

Example 53 2-(S),5-Diamino-pentanoic acid[1-(R)-(5-bromo-thiazol-2-ylcarbamoyl)-3-phenyl-propyl]-amide (Compound61)

2-(S),5-Diamino-pentanoic acid[1-(R)-(5-bromo-thiazol-2-ylcarbamoyl)-3-phenyl-propyl]-amide wasobtained according to Method F.

1H NMR 1.50-1.74 (m, 4H), 1.92-2.09 (m, 2H), 2.53-2.70 (m, 2H),2.76-2.84 (m, 2H), 3.48 (brs, 1H), 4.54 (brs, 1H), 7.16-7.29 (m, 6H),7.56 (s, 1H), 8.62 (brs, 1H)

MS C₁₈H₂₄NO₂S⁷⁸Br (M+H)⁺=454

Example 54 2-(S),5-Diamino-pentanoic acid[1-(R)-(benzothiazol-2-ylcarbamoyl)-3-phenyl-propyl]-amide (Compound 63)

2-(S),5-Diamino-pentanoic acid[1-(R)-(benzothiazol-2-ylcarbamoyl)-3-phenyl-propyl]-amide was obtainedaccording to Method F.

1H NMR 1.49-1.56 (m, 1H), 1.61-1.73 (m, 3H), 1.93-2.03 (m, 1H),2.05-2.15 (m, 1H), 2.56-2.63 (m, 1H), 2.66-2.73 (m, 1H), 2.77-2.83 (m,2H), 3.34 (brs, 1H), 4.56-4.60 (m, 1H), 6.15 (brs, 4H), 7.16-7.32 (m,7H), 7.40-7.45 (m, 1H), 7.71-7.75 (m, 1H), 7.93-7.97 (m, 1H), 8.46 (brs,1H)

MS C₂₂H₂₇N₅O₂S (M+H)⁺=426

Example 55 2-(S)-Amino-4-phenyl-N-quinolin-3-yl-butyramide (Compound 67)

2-(S)-Amino-4-phenyl-N-quinolin-3-yl-butyramide was obtained accordingto Method Ha.

¹H NMR 1.76-1.85 (m, 1H), 1.99-2.08 (m, 1H), 2.65-2.81 (m, 2H), 3.44(dd, J=8 Hz, J=5 Hz, 1H), 7.15-7.31 (m, 6H), 7.54-7.58 (m, 1H),7.62-7.66 (m, 1H), 7.91-7.96 (m, 2H), 8.75 (d, J=2 Hz, 1H), 8.99 (d, J=2Hz, 1H)

MS C₁₉H₁₉N₃O (EI) M⁺=305

Example 562-(S),4-Diamino-N-[3-phenyl-1-(R)-(quinolin-6-ylcarbamoyl)-propyl]-butyramide(Compound 68)

2-(S),4-Diamino-N-[3-phenyl-1-(R)-(quinolin-6-ylcarbamoyl)-propyl]-butyramidewas obtained according to Method Ha.

1H NMR 1.76-1.78 (m, 1H), 1.94-2.04 (m, 2H), 2.06-2.17 (m, 1H),2.58-2.75 (m, 2H), 2.93 (t, J=8 Hz, 2H), 3.61 (brs, 1H), 4.56 (brs, 1H),6.20 (brs, 4H), 7.17-7.31 (m, 5H), 7.49 (dd J=8 Hz, J=4 Hz, 1H), 7.84(dd, J=9 Hz, J=2 Hz, 1H), 7.98 (d, J=9 Hz, 1H), 8.61 (brs, 1H), 8.79(dd, J=6 Hz, J=2 Hz 1H), 10.51 (s, 1H)

MS C₂₃H₂₇N₅O₂ (M+H)⁺=406

Biological Data Example 57 Effect of Stereochemistry on EPI Activity

This example shows the initial microbiological evaluation of the effluxpump inhibitory (EPI) activity of four stereoisomeric compounds (1through 4).

EPI activity was recorded as concentration of an EPI compound that isnecessary to increase susceptibility to levofloxacin of the strain of P.aeruginosa, PAM1723, overexpressing the MexAB-OprM efflux pumpeight-fold. The levofloxacin potentiating activity of the test compoundswas assessed by the checkerboard assay (Antimicrobial Combinations,Antibiotics in Laboratory Medicine, Ed. Victor Lorian, M.D., Fourthedition, 1996, pp 333-338, which is incorporated herein by reference inits entirety) using a broth microdilution method performed asrecommended by the NCCLS (National Committee for Clinical LaboratoryStandards (NCCLS), 1997, Methods for Dilution of AntimicrobialSusceptibility Tests for Bacteria That Grow Aerobically, Fourth Edition;Approved Standard. NCCLS Document M7-A4, Vol 17 No. 2, which isincorporated herein by reference in its entirety). In this assay,multiple dilutions of two drugs, namely an EPI and levofloxacin, weretested, alone and in combination, at concentrations equal to, above andbelow their respective minimal inhibitory concentrations (MICs). All EPIcompounds were readily soluble in water and stock solutions wereprepared at a final concentration of 10 mg/ml. Stock solutions werefurther diluted, according to the needs of the particular assay, inMueller Hinton Broth (MHB). Stock solution was stored at −80° C.

The checkerboard assay was performed in microtiter plates. Levofloxacinwas diluted in the x axis, each column containing a single concentrationof levofloxacin. EPIs were diluted in the y axis, each row containing anequal concentration of an EPI. The result of these manipulations wasthat each well of the microtiter plate contains a unique combination ofconcentrations of the two agents. The assay was performed in MHB with afinal bacterial inoculum of 5×105 CFU/ml (from an early-log phaseculture). Microtiter plates were incubated during 20 h at 35° C. andwere read using a microtiterplate reader (Molecular Devices) at 650 nmas well as visual observation using a microtiter plate-reading mirror.The MIC was defined as the lowest concentration of antibiotics, withinthe combination, at which the visible growth of the organism wascompletely inhibited.

TABLE 1 Levofloxacin potentiation by the EPI compounds Antibiotic MIC(μg/l) in the presence of EPI (μg/ml) Compound 0 0.625 1.25 2.5 5 10 2040 80 MPC₈ (μg/l) 1 2 2 1 0.5 0.06 0.03 0.015 0.008 10 2 2 2 2 2 1 0.1250.06 0.03 20 3 2 2 1 0.25 0.03 0.015 0.015 0.015 10 4 2 2 2 2 0.5 0.060.06 0.03 20

The experiment depected in Table 1 demonstrates that all four compoundshave similar levofloxacin potentiating activity (MPC₈ of 10 to 20 μg/ml)against the MexAB-OprM over-producing strain of P. aeruginosa.

The following examples describe in vitro stability of compound 3 incomparison with its three stereoisomer analogs.

Example 58 In Vitro Stability

To test the stability of the compounds in vitro, samples were preparedand analyzed by LC/MS/MS according to the following procedure.

Homogenization: A chunk of fresh tissue was weighed and mixed withsaline at 1:1 ratio (w/v) in a 12×75 mm polypropylene round bottom tube.The mixture was homogenized with a Polytron homogenizer (on ice, 3×10seconds with 10 second intervals at the setting of 7).

Incubation and TCA-precipitation: An appropriate amount of a tissuehomogenate was pre-warmed at 37° C. for 2 minutes in an Eppendorf tube.The compound to be tested was added to the tube to a final concentrationof 2 μg/ml, mixed, and a 60 μl aliquot was immediately taken (as 0 hoursample) and mixed with 120 μl of 4% TCA (trichloroacetic acid) in anEppendorf tube. The temperature of the tube containing the rest of thetissue sample was returned to 37° C. for incubation. Later, samples weretaken as scheduled and mixed with TCA as described above. Plasma andlavage samples were directly incubated with the compounds withoutaddition of saline and homogenization.

LC/MS/MS sample vial preparation: TCA-precipitated sample tubes werevortexed for 30 seconds, centrifuged in a microfuge at top speed (13.2 krpm) for 5 minutes. The supernatant was collected. The pH wasneutralized to about 3.5 with ammonium acetate buffer. The sample (45μl) was transferred to a glass LC/MS/MS vial (Kimble, 11 mm, 1.5 ml) and5 μl of an internal standard was added to a final concentration of 500ng/ml. LC/MS/MS analysis was performed in Chemistry Department of SanDiego State University.

LC/MS/MS protocol: Thermo-Finnigan TSQ Quantum triple sector massspectrometer with electrospray (ESI) ionization was used to analyze thesamples. The operation conditions were: Mobile phase of pH 3.00water/formic acid:acetonitrile-methanol (45%:5%:50% by volume);Thermo-Keystone Betabasic C-18 column (2.1 mm×50 mm) operated at 0.100mL/min.

Stability in Rat Serum and Tissue Homogenates.

As demonstrated by the data depicted in FIG. 1A, compound 1 was highlystable in all of the tissues tests. After 16 hrs of incubation, therewas still 70-80% of the compound left in the reaction tubes.

As demonstrated by the data depicted in FIG. 1B, compound 2 in serum wasintermediately stable. After 1.5 hr of incubation there was still abouthalf of the compound left, but the compound was almost all gone after 4hr. This compound was, however very unstable in kidney and liver. Hardlyany was left after the first 40 minutes of incubation.

As demonstrated by the data depicted in FIG. 1C, compound 3 was at leastas stable as 1 in rat serum. However, this compound appeared to be moreunstable than 1 in kidney and liver.

Example 59 Stability in Tissues at Different pH

Many proteases responsible for degrading EPI compounds are located inlysozymes. Lysozymes typically have an acidic pH (about 4.8-5), whereasthe pH in tissue homogenates that were prepared was higher (kidney, pH6.8; liver, pH 6.6; and lung, pH 7.5). To test the effect of tissuehomogenates closer to physiological conditions, the pH was lowered toabout 6 by adding acetic acid to the tissue homogenates and compoundstability was tested. FIG. 2 depicts the stability of compounds 1 and 3at different pH. The data demonstrate that, while stability of compound1 in different tissues was essentially unchanged, that of compound 3exhibited significant differences. At pH 6, compound 3 was degradedfaster than at no pH change. The pH-dependent degradation of compound 3was especially evident in kidney homogenates.

FIG. 3 depicts the level of compound 3 and the expected degradationproduct of compound 3 (compound 67) as a function of time. Concomitantwith the disappearance of compound 3, the degradation product 67increased in rat kidney homogenates.

Example 60 Stability in Tissue Homogenate Supernatant

FIG. 4 is a bar graph depicting the stability of compound 3 in thesupernatant and pellet of kidney tissue homogenates. Greater instabilityof compound 3 was observed in the supernatant of the kidney tissuehomogenates obtained after 5 min. centrifugation in the microcentrifuge.This result indicates that enzymes which are responsible for thedegradation of 3 are soluble as opposed to membrane bound.

FIG. 5 compares the stability of compounds 1, 3, and 4 in rat kidneytissue homogenates. The supernatant instability was selective tocompound 3: neither compound 1 nor compound 4 show any instability atthe same conditions.

Moreover, it appears that the level of degradation observed in thepresence of high-speed supernatant (80K) was much higher that oneobserved in the presence of the non-fractionated homogenate. This resultsupports that membranes might contain inhibitors of peptidases involvedin selective degradation of compound 3.

Example 61 Stability of Compound 3 in Rabbit Tissue Homogenates

FIG. 6 compares the stability of compounds 1 and 3 in fractionatedrabbit kidney tissue homogenates. Similar instability of compound 3 ascompared to compound 1 was observed in the supernatant of rabbit kidneyhomogenates.

Greater instability is observed in the 80K supernatant as compared to18K supernatant.

Example 62 Time Dependence of EPI Activity

Stability of compounds was also evaluated using a bioassay. The testcompound at 320 μg/ml was treated with rabbit kidney supernatant for 0,0.5, 1 and 2 hours. 120 μl was taken at designated times, heated at 80°C. to inactivate the enzyme, centrifuged and used in levofloxacinpotentiation assay to determine whether or not their levofloxacinpotentiation activity is reduced after the treatment. Levofloxacinpotentiation activity was defined as the amount of a test compound,which is essential to decrease MIC of the strain PAM1723 of P.aeruginosa eight-fold (MPC₈). In order to establish MPC₈, the MIC of thetest compound for PAM1723 (MIC=2 μg/ml) grown in the presence of 0.25μg/ml of levofloxacin was determined.

TABLE 2 Evaluation of tissue instability of EPIs using bioassay. EPIMPC₈ after incubation (h) with rabbit kidney supernatant at 37° C.Saline Compound (2 hours) 0 0.5 1 2 1 5 10 10 10 10 3 5 20 40 81 81 81means that activity is >80 μg/ml

The bioassay indicated that compound 3 but not compound 1 demonstratestime-dependent reduction of the EPI activity after treatment withsupernatant of rabbit kidney homogenate (Table 2).

Example 63 Stability of Compound 3 in Human Serum and Tissue Homogenates

The pattern of in vitro stability of compound 3 (stable in serum butunstable in tissues) observed in rodents was also reproduced using humankidney tissues or serum. FIG. 7 compares the in vitro stability ofcompounds 1 and 3 in human kidney tissue preparations. Time-dependentdegradation of compound 3 but not compound 1 was demonstrated wheneither compound was treated with homogenates prepared from frozencadaverous kidney. Moreover, the degradation of compound 3 was strongerat lower pH, indicating that the acidic pH is essential for optimalhydrolytic activity.

FIG. 8 depicts formation of the metabolite, compound 67, in human kidneytissue. Concomitant with the disappearance of compound 3, generation ofthe expected metabolite of the presumed intralysosomal degradation,compound 67, was detected using human kidney.

Example 64 Stability of Compound 3 in Human Serum

FIG. 9 compares the stability of compounds 1 and 3 in human serum as afunction of time. Both compound 1 and compound 3 were stable after 18hours of incubation with human serum. This result supports that thedegradation of compound 3 is selective for the intracellular enzymeswith low pH being optimum, such as those found in lysosomes.

Example 65 Stability Bioassay

Stability in rat and human serum was also evaluated using a bioassay.320 μg/ml of the test compound was treated with 50% serum for 4 hours.Next, the MIC of a test compound for PAM1723 (MIC=2 μg/ml) grown in thepresence of 0.25 μg/ml of levofloxacin was determined (MPC8), andcompared with that of a compound treated with saline for 4 hours.Compound MC-207110 (L-phenylalanine-L-ornithine-a-naphtylamide) which isunstable in both rat and human serum, was used as a positive control(Table 3).

TABLE 3 Evaluation of serum stability of EPIs using bioassay. EPI MPC₈after 4 hours at 37° C. Frozen Frosen Rat Human Compound Saline serumserum MC-207110 2.5 >80 >80 3 5 5 5

Example 66 Stability in P. aeruginosa

Overnight cultures of two different strains of P. aeruginosaoverexpressing (strain PAM1723) or lacking (PAM1626) efflux pumps wereincubated with compounds 1 or 3 at 50 μg/ml for various times. Compoundswere extracted using 4% TCA and analysed by LC/MC/MC. The resultspresented on FIG. 10 demonstrated that both compounds 1 and 3 are stablein cultures of tested bacterial strains. This data supports that EPIcompounds with hydrolytic instability are not degraded by bacterialcells.

Example 67 Bioassays of Several EPI Compounds

Bioassays as described in Example 62 and Example 65 were used toevaluate the stability of various compounds in serum and rabbit kidneyhomogenates. Decreased levofloxacin potentiation activity after thetreatments is indicative of enzymatic instability of compounds (Table4).

TABLE 4 EPI Activity and Stability of Representative Compounds in Serumand Kidney Tissues Evaluated Using Bioassay. EPI MPC₈ (μg/ml) after EPIMPC₈ (μg/ml) after incubation (h) with rabbit 4 hours at 37° C. kidneysupernatant at 37° C. Frozen Rat Frosen Human Saline Compound # Salineserum serum (2 hours) 0 0.5 1 2 1 5 5 5 5 10 10 10 10 3 5 5 5 5 2040 >80 >80 6 2.5 2.5 2.5 5 10 20 40 >80 7 10 10 10 10 40 80 >80 >80 82.5 2.5 2.5 2.5 10 20 80 >80 9 5 >80 5 5 40 >80 >80 >80 10 5 10 5 5 2020 80 >80 11 2.5 2.5 2.5 2.5 10 20 >80 >80 13 <2.5 <2.5 <2.5 <2.540 >40 >40 >40 16 10 10 20 10 40 80 >80 >80 17 5 5 5 5 10 >80 >80 >80 1880 >80 >80 80 >80 >80 >80 >80 19 40 >80 >80 80 >80 >80 >80 >80 21 <1.25<1.25 <1.25 <1.25 5 10 20 80 23 10 10 20 10 40 40 >80 >80 25 2.5 10 10 580 >80 >80 >80 26 2.5 2.5 2.5 2.5 80 >80 >80 >80 28 5 >40 105 >40 >40 >40 >40 29 10 20 10 10 40 40 >80 >80 30 5 5 40 5 20 20 80 >8031 10 10 10 10 20 40 >80 >80 32 5 10 10 10 20 80 >80 >80 34 40 >80 >8040 >80 >80 >80 >80 36 10 >80 20 10 >80 >80 >80 >80 37 10 10 10 10 2080 >80 >80 39 10 >40 20 10 >40 >40 >40 >40 40 2.5 10 2.5 5 20 40 >80 >8041 2.5 2.5 2.5 2.5 10 20 40 >40 46 1.25 1.25 1.25 1.25 20 40 >40 >40These compounds demonstrate serum stability but instability in thesupernatant of rabbit kidney homogenates. Several compounds, which areunstable in rat kidney homogenates are also unstable in rat serum (e.g.,compounds 9, 18, 19, 28, 34, 36, and 39).

Several examples below provide in vivo characterization of the selectedEPI compounds.

Example 68 Rat Serum Pharmacokinetics After 1 min IV BolusAdministration

Rat serum pharmacokinetics of compounds 1, 2, 3 and 4 was evaluatedafter IV bolus administration (FIG. 1). FIG. 11 depicts the serumconcentrations as a function of time. The PK profiles of compounds 1, 2and 4 were very similar while the compound 2 levels decreased morerapidly faster. These data support the instability of compound 2 inserum as compared with serum stability of compounds 1, 2 and 4.

Example 69 Rat Serum Pharmacokinetics After 2-hour IV Infusion

Rat serum pharmacokinetics of compounds 1, 3, 7, and 21 was evaluatedafter 2-hour IV infusion of 1 mg/ml EPI solution in 0.9% saline. Totalinfused dose was 10 mg/kg. FIG. 12 depicts the serum concentrations as afunction of time. A two-compartment model was used to fit the data andcalculate PK parameters. All compounds, regardless of their stability intissue homogenates, showed a similar PK profile: rapid alpha-eliminationwith a longer beta-phase. Compound 7, which was unstable in tissuehomogenates, had the best serum PK profile.

TABLE 5 Pharmacokinetics after IV infusion. Compound CL (L/hr/kg) 1 1.443 1.4 7 0.39 21 0.97

Example 70 Tissue Levels of 1, 2, 3 and 4 After IV Bolus Administration

Tissue levels of compounds 1, 2, 3 and 4 were evaluated in kidney, liverand lung six-hours after 8.4 mg/kg IV bolus administration. FIG. 13depicts the levels as a function of time. At 6 hours after IV injection,compound 2 levels fell below the detectable level in all the tissuestested and compound 3 was also present at very low level. On the otherhand, compounds 1 and 4 were present at relatively high levels,especially in kidney.

This experiment indicated that there is a correlation between theinstability in tissue homogenates and tissue levels of the testcompounds: 1 and 4 were shown to be stable in tissue homogenates and thesame compounds are found in tissues at much higher levels than theunstable compounds 2 and 3.

Example 71 Tissue Levels of Selected EPIs After Two-Hour IV Infusion

Levels of EPIs in various tissues were evaluated after 2-hour IVinfusion in rats. The rats received 10 mg/kg IV infusion through FVC.Animals were divided into 2 groups: group 1 was sacrificed immediatelyafter the end of infusion and group 2, 2h or 4h after the end ofinfusion. Tissues from each animal were collected, homogenized andcompounds were extracted. Tissues were mixed with saline at 1:1 ratio(v/w) in 12×75 mm polypropylene round bottom tubes. The mixtures werehomogenized with a Polytron homogenizer (on ice, 3×10-15 seconds with 30seconds intervals at the setting of 7). 50 μl of homogenized tissue wereimmediately transferred to an Eppendorf tube and 2-volumes of 4% TCAwere added. After vortexing for 30 seconds, the tubes were centrifugedin a microfuge at top speed (13.2 k rpm) for minutes. The supernatantwas collected, and 45 μl of the supernatant was transferred to a glassLC/MS/MS vial (Kimble, 11 mm, 1.5 ml) for analysis, which alloweddetermination of tissue levels of EPIs at the end of the infusion and 2hor 4h later.

TABLE 5 Tisue Levels of compound 1 after 2-hour Infusion in RatConcentration Concentration (μg/ml) Tissue (μg/ml) at 2 h at 4 h 2 h/4 hRatio Kidney 49.31 29.64 1.66 Liver 10.55 10.62 0.99 Lung 6.50 4.18 1.55Muscle 4.54 2.86 1.59 Brain 0.18 0.13 1.34 Pancreas 5.40 4.05 1.34Thymus 3.41 2.76 1.23 Spleen 5.01 3.71 1.35 Heart 2.18 2.34 0.93Testicles 0.58 0.28 2.04

High levels of compound 1 was observed in several tissues includingkidney, liver, and lung. No significant changes in the level of compound1 were observed two-hours after the end of infusion (Table 5).

TABLE 6 Tisue Levels of compound 3 after 2-hour Infusion in RatConcentration Concentration Tissue (μg/ml) at 2 h (μg/ml) at 4 h 2 h/4 hRatio Kidney 12.21 1.68 7.28 Liver 1.33 0.83 1.60 Lung 4.27 1.91 2.23Muscle 2.18 1.14 1.91 Brain 0.07 0.21 0.35 Pancreas 1.11 0.38 2.91Thymus 2.46 1.46 1.69 Spleen 4.41 0.72 6.16 Heart 2.26 0.47 4.82Testicles 0.32 0.06 5.14

Lower levels of compound 3 as compared to 1 were observed in severaltissues including kidney, liver and lung. Significant changes in thelevel of compound 3 were observed two-hours after the end of infusion(Table 6).

TABLE 7 Tisue Levels of 7 after 2-hour Infusion in Rat ConcentrationConcentration Tissue (μg/ml) at 2 h (μg/ml) at 6 h 2 h/4 h Ratio Kidney55.36 0.96 57.37 Liver 7.96 0.27 29.85 Lung 13.04 3.17 4.11

While relatively high levels of compound 7 were observed in tissues atthe end of 2-hour infusion, this compound cleared from tissuesrelatively fast, presumably due to selective intralysosomal degradation(Table 7).

TABLE 8 Tisue Levels of 21 after 2-hour Infusion in Rat ConcentrationConcentration Concentration 2 h/6 h Tissue (μg/ml) at 2 h (μg/ml) at 4 h(μg/ml) at 6 h Ratio Kidney 58.71 20.76 2.92 20.10 Liver 5.95 2.32 0.2030.21 Lung 13.39 NA 3.90 3.43 Muscle 4.75 1.36 1.21 3.94 Brain 0.49 0.470.03 16.37 Pancreas 9.71 1.58 0.47 20.73 Thymus 7.25 6.00 3.59 2.02Spleen 7.66 4.44 2.21 3.46 Heart 5.25 1.76 0.72 7.33 Testicles 0.70 0.330.08 8.32

Slower clearance of compound 21 was observed 2 hours after the end ofthe infusion. Significantly less compound was detected in tissues 2hours later, indicating tissue-specific degradation of compound 21(Table 8).

Example 72 Tissue Levels of Compound 3 After 5 Day Repeated Dosing inRat

The decreased tissue accumulation of compound 3 vis-a-vis 1 wasconfirmed in a multi-dose experiment. Rats received 20 mg/kg of eithercompound twice daily for 5 days. Animal were sacrifised 6 hours afterthe last dose and levels of compounds in kidneys, liver and lung weredetermined. FIG. 14 depicts the measured tissue levels. Approximately100-fold, 60-fold, and 10-fold less compound 3 accumulated in kidney,liver and lung respectively, as compared to compound 1.

Example 73 Tissue Levels of Compound 67 After IV Administration in Rats

Compound 67, which is the expected metabolite of proteolyticintracellular degradation of compound 3, was detected in rat tissuesafter IV infusion, confirming the tissue-specific degradation ofcompound 3 (Tables 9A, B).

TABLE 9A Tissue levels of 67 after 6-Hours or 5 Day Repeated Dosing inRats. Level of 67 6-hour after IV (8.4 mg/kg) Kidneys Liver Lung Average(mg/kg) 0.28 0.09 1.09 stand'd dev. 0.03 0.04 0.05

TABLE 9A Tissue levels of 67 after 6-Hours or 5 Day Repeated Dosing inRats. 6-hour after 5-day IV Level of 67 (20 mg/kg/BID) kidney liver lungAverage (mg/kg) 0.11 0.04 0.34 stand'd dev. 0.00 0.06

Example 74 Tissue Levels of Compounds 1 and 3 After IP Administration inMice

Levels of compounds 1 and 3 were evaluated in kidney, liver and lungsix-hours after 10 mg/kg IP bolus administration in mice. FIG. 15depicts the resulting tissue levels. The results show that the presumedintracellular hydrolytic degradation also occurs in mice. Thus, compound3 and other compounds can be used in mouse efficacy models of P.aeruginosa infection.

Example 75 Tissue levels of Compound 3 After Administration of AcylatedForms

The present example indicates that compound 5, in which compound 3 wasacylated at its alfa-NH₂, was readily metabolized in serum to producecompound 3. Rats were injected with compound 5 (8.4 mg/kg) and serumlevels of the parent compound 3 were evaluated after extraction. Theserum levels as a function of time are depicted in FIG. 16.

Compound 43, in which compound 3 was acylated at both of its free aminefunctionalities is also readily metabolized in serum to produce compound3. Rats were infused for two hours with compound 43 at 10 mg/kg andserum levels of the parent compound 3 were evaluated after extraction.FIG. 17 depicts the serum levels of compound 3 as a function of time.

Example 76 Acute Toxicity of Acylated Compound 3

The present example demonstrates that the substitution of a single orboth free amines functionalities of compound 3 with the acyl groupreduced 2 to 8-fold acute toxicity of the parent compound. Acutetoxicity was measured as minimal lethal dose (MLD). MLD is defined as aminimal concentration of a compound that has lethal effect for at leastone mouse from the group of three mice after IV bolus injection into themouse tail vein. Compounds 5, 34, and 43 did not have levofloxacinpotentiating activity in vitro at concentrations up to 80 μg/ml.

TABLE 10 Minimal lethal doses of selected compounds after bolusadministration in mice Compound MLD (mg/kg) 3 25 5 75 34 50 43 150

Several examples below demonstrate efficacy of selected EPI compounds.6-8 week old Swiss Webster male mice were used for the efficacyexperiments.

Example 77 Efficacy of Compound 1 in Mouse Sepsis Models of PA InfectionInoculum Preparation

P. aeruginosa strain PAM1032 overexpressing MexAB-OprM efflux pump wasgrown overnight in Mueller-Hinton Broth (MHB). The next day, theovernight culture was diluted in fresh MHV and was allowed to re-grow to˜108 CFU/mL (OD600˜0.3). The culture was spun down, washed twice withphosphate buffered saline (PBS) and re-suspend in PBS to ˜1.2×107 CFU/mL(OD=0.12). Next, this culture was mixed 1:1 with 14% hog-gastric mucinin PBS to give a final inoculum of 6×106 CFU/mL.

Infection and Treatment

Each group of animals was infected with 0.5 mL of inoculum via theintraperitoneal (IP) route and then was immediately treated with 0.2 mLof levofloxacin or PBS via the subcutaneous (SQ) route, and next with0.2 mL EPI via the IP route. Two hours after the first treatment, theanimals were treated again with 0.2 mL of levofloxacin or PBS via the SQroute and 0.2 mL of EPI via the IP route. After 24 hours, the proportionof surviving animals in each treatment group was calculated and ED50 wasdetermined.

Treatment Groups to Determine ED₅₀

Twelve groups of 5 mice were used to determine the effect of each fixedconcentration of EPI compound on ED50 of levofloxacin. The effect of theEPI at 50 mg/kg was determined according to the following experimentaldesign:

1 Levofloxacin 200 mg/kg (100 mg/kg × 2) 2 Levofloxacin 200 mg/kg plusEPI 50 mg/kg (100 and 25 mg/kg × 2 respectively) 3 Levofloxacin 100mg/kg (50 mg/kg × 2) 4 Levofloxacin 100 mg/kg plus EPI 50 mg/kg (50 and25 mg/kg × 2 respectively) 5 Levofloxacin 50 mg/kg (25 mg/kg × 2) 6Levofloxacin 50 mg/kg plus EPI 50 mg/kg (25 and 25 mg/kg × 2respectively) 7 Levofloxacin 25 mg/kg (12.5 mg/kg × 2) 8 Levofloxacin 25mg/kg plus EPI 50 mg/kg (12.5 and 25 mg/kg × 2 respectively) 9Levofloxacin 12.5 mg/kg (6.25 mg/kg × 2) 10 Levofloxacin 12.5 mg/kg plusEPI 50 mg/kg (6.25 and 25 mg/kg × 2 respectively) 11 PBS plus EPI 50mg/kg (6.25 and 25 mg/kg × 2 respectively) 12 Virulence control (PBStreatment)

The percent survival at 24 hours post infection for levofloxacin dosesof 0, 12.5, 25, 50, 100, and 200 mg/kg alone, and for levofloxacin dosesof 0, 12.5, 25, 50, 100, and 200 mg/kg with 50 mg/kg of MP-001,001 areplotted in FIG. 18, In this study, 4-fold decrease of ED₅₀ oflevofloxacin was observed in the presence of compound 1 at 50 mg/kg.

Efficacy of 3 in Mouse Sepsis Model of PA Infection.

Efficacy of compound 3 in the Mouse Sepsis Model was evaluated as in forcompound 1. Several concentrations of compound 3 were used to determinetheir effect on the ED₅₀ of levofloxacin. In these studies, adose-dependent decrease in ED₅₀ (up to 4-fold) was observed in thepresence of increasing concentrations of compound 3. The survival ratesare in FIG. 19.

Example 78 Efficacy EPIs in Neutropenic Mice Lung Model of PA Infection

Neutropenia was induced by cyclophosphamide 100 mg/kg IP on days 3 and 1prior to infection. The strain 29A2 of P. aeruginosa overexpressingMexAB-OprM efflux pump was grown overnight in Mueller-Hinton Broth(MHB). The next day overnight culture was diluted in fresh MHB and wasallowed to re-grow to ˜10⁸ CFU/mL (OD600˜0.3). The culture was spundown, washed twice with phosphate buffered saline (PBS) and re-suspendedin PBS to ˜4×10⁷ CFU/mL. 50 μl of inoculum was used for intratrachealinstillation of mice, which were anesthetized with sevoflourane/oxygen.Mice were treated with Levofloxacin SQ (Total dose is 30 mg/kg) and/orEPI IP (Total dose was 100 mg/kg) at 0 and 2 hours post infection. Fourmice from each treatment group were euthanized at various times afterthe start of treatment. The lungs were removed aseptically, homogenizedin saline, and immediately plated to determine bacterial load (CFU/ml)in infected lungs. P. aeruginosa is plotted as a function of time inFIG. 20. No difference in growth of P. aeruginosa was observed witheither no addition (0/0) or with 100 mg/kg of compound 1 alone (0/100).Levofloxacin at 30 mg/kg (30/0) had static effect while addition ofcompound 1 to levo significantly decreased bacterial load in theinfected lungs (30/100) indicating potentiating effect in vivo.

The effect of compound 3 in the same mouse model was also studied. P.aeruginosa levels were plotted after administration of compound 3 at 100mg/kg (FIG. 21A) and 40 mg/kg (FIG. 21B). Compound 3, which was moreunstable than compound 1 in lung tissue homogenates, still had thedose-dependent efficacy in levofloxacin potentiation.

In summary, both compounds 1 and 3 had similar efficacy in a pneumoniamodel of P. aeruginosa infection despite their differential stability inlung cells.

Efficacy of Compound 5 in Neutrapenic Mice Lung Model of PA Infection.

The effect of compound 3 in the same mouse model was also studied. FIG.22 is a graph of P. aeruginosa levels after administration of compound5. Since compound 5 appears to be readily converted to compound 3 inserum, it was, as expected, efficacious in animal models of P.aeruginosa infection. Compound 5 (which is devoid of levofloxacinpotentiating activity) was efficacious in a Neutropenic Mice Lung Modelof P. aeruginosa Infection.

Example 79 Efficacy of EPIs in Neutrapenic Mice Thigh Model of PAInfection

Efficacy of the EPIs was evaluated in a Neutropenic Mice Thigh Model ofP. aeruginosa Infection. Neutropenia was induced by cyclophosphamide 100mg/kg IP on days 3 and 1 prior to infection. P. aeruginosa strainPAM1723 overexpressing MexAB-OprM efflux pump was grown overnight inMueller-Hinton Broth (MHB). The next day, the overnight culture wasdiluted in fresh MHB and was allowed to re-grow to ˜10⁸ CFU/mL(OD600˜0.3). The culture was spun down, washed twice with phosphatebuffered saline (PBS) and re-suspend in PBS to ˜4×10⁶ CFU/mL. Next, thisculture was mixed 1:1 with 14% hog-gastric mucin in PBS to give a finalinoculum of 2×10⁶ CFU/mL. Infection was initiated with injection of 0.1mL of inoculum (˜2×10⁵ CFU/thigh) directly into both thighs of eachmouse. Two mice from each treatment group were euthanized at varioustimes after the start of treatment. The thighs were removed aseptically,homogenized in saline, and immediately plated to determine bacterialload (CFU/ml). Treatment was initiated 2 hours post infection.Levofloxacin (30 mg/kg) and EPIs (60 mg/kg) were given SQ and IP,respectively in 0.2 ml of saline.

In the first experiment, levofloxacin was given at 30 mg/kg and compound1 was given at 60 mg/kg 2 hours post-infection. FIG. 23 depicts thebacterial load as a function of time. Compound 1 significantly enhancedactivity of levofloxacin as evident by the decreased load of bacteria atthe site of infection in the presence of the EPI as compared tolevofloxacin alone.

In the second experiment, levofloxacin was given at 30 mg/kg andcompound 3 or compound 43 was given at 60 mg/kg 2 hours post-infection.Compound 43 is a derivative of compound 3 in which both primary aminesare converted into L-aspartatamines. FIG. 24 depicts the bacterial loadas a function of time. Compound 43 did not have in vitro levofloxacinpotentiating activity. However, both compounds 3 and 43 potentiatedlevofloxacin in vivo as demonstrated in neutropenic mouse thigh model ofinfection.

Efficacy of Compound 21 in Neutrapenic Mice Thigh Model of PA Infection.

Compound 21 (a potent compound that was unstable in tissue homogenatesand does not accumulate in rat tissues), was also tested in the thighmodel as above. FIG. 25 depicts the resulting bacterial load as afunction of time. Compound 21 significantly enhanced activity oflevofloxacin as evident by the decreased load of bacteria at the site ofinfection in the presence of the EPI as compared to levofloxacin alone.

All references cited herein are incorporated herein by reference intheir entirety. To the extent publications and patents or patentapplications incorporated by reference contradict the disclosurecontained in the specification, the specification is intended tosupersede and/or take precedence over any such contradictory material.

All numbers expressing quantities of ingredients, reaction conditions,and so forth used in the specification and claims are to be understoodas being modified in all instances by the term “about.” Accordingly,unless indicated to the contrary, the numerical parameters set forth inthe specification and attached claims are approximations that may varydepending upon the desired properties sought to be obtained by thepresent invention. At the very least, and not as an attempt to limit theapplication of the doctrine of equivalents to the scope of the claims,each numerical parameter should be construed in light of the number ofsignificant digits and ordinary rounding approaches.

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only and are not meant to be limiting in anyway. It is intended that the specification and examples be considered asexemplary only.

1. A compound having the structure of Formula (III):

wherein: R₁ is:

X₁ is selected from the group consisting of —O— and —S—; m₁ is aninteger from 0 to 4; n₁ is an integer from 0 to 1; R₁₁ is selected fromthe group consisting of —NH₂, —NH—CH(═NH), —NH—C(CH₃)(═NH), —CH(═NH)NH₂,and —NH—C(═NH)NH₂; R₃ is selected from the group consisting of:

X₃ is selected from the group consisting of —CH₂—, —C(CH₃)₂—, —O—, and—S—; m₃ is an integer from 1 to 2; n₃ is an integer from 0 to 2; R₃₁ isselected from the group consisting of C₁₋₁₀ alkyl, C₁₋₁₀ alkenyl, C₁₋₁₀alkynyl, and C₁₋₁₀ cycloalkyl; R₄ is selected from the group consistingof hydrogen, optionally substituted C₁₋₆ alkyl, and optionallysubstituted C₃₋₇ cycloalkyl; Ar is an optionally substituted phenyl, R₅is selected from the group consisting of:

each optionally substituted with methyl, ethyl, n-propyl, isopropyl,cyclopropyl, tert-butyl, hydroxyl, methoxyl, ethoxyl, hydroxymethyl,trifluoromethyl, trifluoromethoxyl, or halogen moieties, and

wherein: each A₅ is separately selected from the group consisting of═CH— and ═N—, with the proviso that each A₅ containing ring contains nomore than four ═N— groups; each B₅ is separately selected from the groupconsisting of —C═, —N═, —O—, —S—, —NH—, and —N(R₆)—, with the provisothat each B₅ containing ring contains no more than three heteroatoms; R₆is selected from the group consisting of hydrogen, C₁₋₆ alkyl, and C₃₋₆cycloalkyl; R₅₁ is selected from the group consisting of hydrogen,methyl, ethyl, and cyclopropyl; m₅ is an integer from 1 to 3; n₅ is aninteger from 0 to 2; p₅ is an integer from 0 to 4; and any amino groupspresent in R₁ are optionally acylated with an amino acid having an(S)-configuration selected from aspartic acid and glutamic acid.
 2. Thecompound of claim 1, wherein R₃ is selected from the group consistingof:

wherein: R₃₁ is selected from the group consisting of ethyl, propyl,isopropyl, isobutyl, tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl,and cyclohexyl; and m₃ is an integer from 1 to
 2. 3. The compound ofclaim 1, wherein —N(R₄)(R₅) is selected from the group consisting of:


4. The compound of claim 3, selected from the group consisting of:


5. A pharmaceutical composition, comprising a compound of 1 in an amounteffective to inhibit an efflux pump of a microbe.
 6. A pharmaceuticalcomposition, comprising a compound of 1 in combination with anantimicrobial agent.
 7. The compound of claim 1, wherein: n1 is 0; m1 isan integer from 2 to 4; and R₁₁ is —NH₂.
 8. The compound of claim 1,wherein: R₃₁ is selected from the group consisting of C₁₋₁₀ alkyl andC₃₋₁₀ cycloalkyl; X₃ is —CH₂—; and n3 is an integer from 0 to
 1. 9. Thecompound of claim 3, wherein: n1 is 0; m1 is an integer from 2 to 4; R₁₁is —NH₂; R₃₁ is selected from the group consisting of C₁₋₁₀ alkyl andC₃₋₁₀ cycloalkyl; X₃ is —CH₂—; and n3 is an integer from 0 to
 1. 10. Thecompound of claim 9, wherein R₃ is


11. A compound having the structure of Formula (III):

wherein: R₁ is:

X₁ is selected from the group consisting of —O— and —S—; m₁ is aninteger from 0 to 4; n₁ is an integer from 0 to 1; R₁₁ is selected fromthe group consisting of —NH₂, —NH—CH(═NH), —NH—C(CH₃)(═NH), —CH(═NH)NH₂,and —NH—C(═NH)NH₂; R₃ is selected from the group consisting of:

X₃ is selected from the group consisting of —CH₂—, —C(CH₃)₂— —O—, and—S—; m₃ is an integer from 1 to 2; n₃ is an integer from 0 to 2; R₃₁ isselected from the group consisting of C₁₋₁₀ alkyl, C₁₋₁₀ alkenyl, C₁₋₁₀alkynyl, and C₁₋₁₀ cycloalkyl; R₄ is selected from the group consistingof hydrogen, optionally substituted C₁₋₆ alkyl, and optionallysubstituted C₃₋₇ cycloalkyl; Ar is phenyl substituted with one or moregroups independently selected from the group consisting of heterocyclyl,heteroaryl, chlorine, bromine, iodine, hydroxy, amino, cyano, nitro,alkylamido, acyl, C₁₋₆ alkoxy, C₁₋₆ alkyl, C₁₋₆ hydroxyalkyl, C₁₋₆aminoalkyl, C₁₋₆ alkylamino, alkylsulfenyl, alkylsulfinyl,alkylsulfonyl, sulfamoyl, or trifluoromethyl, and R₅ is

optionally substituted with methyl, ethyl, n-propyl, isopropyl,cyclopropyl, tert-butyl, hydroxyl, methoxyl, ethoxyl, hydroxymethyl,trifluoromethyl, trifluoromethoxyl, or halogen moieties; each A₅ isseparately selected from the group consisting of ═CH— and ═N—, with theproviso that each A₅ containing ring contains no more than four ═N—groups; n₅ is an integer from 0 to 2; and any amino groups present in R₁are optionally acylated with an amino acid having an (S)-configurationselected from aspartic acid and glutamic acid.
 12. The compound of claim11, selected from the group consisting of:


13. The compound of claim 11, selected from the group consisting of:


14. The compound of claim 1, wherein R₄ is hydrogen.
 15. A compoundhaving the structure of Formula (III):

wherein: R₁ is:

X₁ is selected from the group consisting of —O— and —S—; m₁ is aninteger from 0 to 4; n₁ is an integer from 0 to 1; R₁₁ is selected fromthe group consisting of —NH₂, —NH—CH(═NH), —NH—C(CH₃)(═NH), —CH(═NH)NH₂,and —NH—C(═NH)NH₂; R₃ is selected from the group consisting of:

X₃ is selected from the group consisting of —CH₂—, —C(CH₃)₂— —O—, and—S—; m₃ is an integer from 1 to 2; n₃ is an integer from 0 to 2; R₃₁ isselected from the group consisting of C₁₋₁₀ alkyl, C₁₋₁₀ alkenyl, C₁₋₁₀alkynyl, and C₁₋₁₀ cycloalkyl; Ar is an optionally substituted phenyl,R₄ is bound to R₅ to form a five-membered or six-membered heterocyclicring selected from the group consisting of:

any amino groups present in R₁ are optionally acylated with an aminoacid having an (S)-configuration selected from aspartic acid andglutamic acid.
 16. A method of treating a bacterial infection,comprising administering to a subject suffering from said bacterialinfection an amount effective to inhibit an efflux pump of said bacteriaof a compound of claim 1 or
 11. 17. The method of claim 16, wherein thesubject is a human.
 18. The method of claim 16, wherein the bacteria isselected from the group consisting of Pseudomonas aeruginosa,Pseudomonas fluorescens, Pseudomonas acidovorans, Pseudomonasalcaligenes, Pseudomonas putida, Stenotrophomonas maltophilia,Burkholderia cepacia, Aeromonas hydrophilia, Escherichia coli,Citrobacter freundii, Salmonella typhimurium, Salmonella typhi,Salmonella paratyphi, Salmonella enteritidis, Shigella dysenteriae,Shigella flexneri, Shigella sonnei, Enterobacter cloacae, Enterobacteraerogenes, Klebsiella pneumoniae, Klebsiella oxytoca, Serratiamarcescens, Francisella tularensis, Morganella morganii, Proteusmirabilis, Proteus vulgaris, Providencia alcalifaciens, Providenciarettgeri, Providencia stuartii, Acinetobacter calcoaceticus,Acinetobacter haemolyticus, Yersinia enterocolitica, Yersinia pestis,Yersinia pseudotuberculosis, Yersinia intermedia, Bordetella pertussis,Bordetella parapertussis, Bordetella bronchiseptica, Haemophilusinfluenzae, Haemophilus parainfluenzae, Haemophilus haemolyticus,Haemophilus parahaemolyticus, Haemophilus ducreyi, Pasteurellamultocida, Pasteurella haemolytica, Branhamella catarrhalis,Helicobacter pylori, Campylobacter fetus, Campylobacter jejuni,Campylobacter coli, Borrelia burgdorferi, Vibrio cholerae, Vibrioparahaemolyticus, Legionella pneumophila, Listeria monocytogenes,Neisseria gonorrhoeae, Neisseria meningitidis, Kingella, Moraxella,Gardnerella vaginalis, Bacteroides fragilis, Bacteroides distasonis,Bacteroides 3452A homology group, Bacteroides vulgatus, Bacteroidesovalus, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroideseggerthii, and Bacteroides splanchnicus.
 19. A method for inhibitinggrowth of antibacterial-resistant bacteria, comprising contacting thebacteria with a compound of claim 1 or 11 and an antibacterial agent.20. The method of claim 19, wherein the antibacterial agent is selectedfrom the group consisting of a quinolone, aminoglycoside, beta-lactam,coumermycin, chloramphenical, lipopeptide, glycopeptide, glycylcycline,ketolide, macrolide, oxazolidonone, rifamycin, streptogramin, andtetracycline.
 21. The method of claim 20, wherein the antibacterialagent is selected from the group consisting of ciprofloxacin,levofloxacin, moxifloxacin, ofloxacin, gatifloxacin, cinoxacin,gemifloxacin, norfloxacin, lomofloxacin, pefloxacin, garenoxacin,sitafloxacin, and DX-619.
 22. A method for treating a bacterialinfection in a subject, wherein the bacteria causing the infectionexhibit antibiotic resistance through an efflux pump mechanism,comprising: administering to a subject an antibiotic to which saidbacteria are resistant; and administering to said subject a compound ofclaim 1 or 11 in conjunction with said antibiotic.
 23. The method ofclaim 22, wherein the antibiotic is selected from the group consistingof a quinolone, aminoglycoside, beta-lactam, coumermycin,chloramphenical, lipopeptide, glycopeptide, glycylcycline, ketolide,macrolide, oxazolidonone, rifamycin, streptogramin, and tetracycline.24. The method of claim 23, wherein the antibiotic is selected from thegroup consisting of ciprofloxacin, levofloxacin, moxifloxacin,ofloxacin, gatifloxacin, cinoxacin, gemifloxacin, norfloxacin,lomofloxacin, pefloxacin, garenoxacin, sitafloxacin, and DX-619.
 25. Thecompound of claim 11, having the structure: